Methods for identifying a GLP-1 secretagogue

ABSTRACT

The present invention concerns combination of an amount of a BRS-3 agonist with an amount of a dipeptidyl peptidase IV (DPP-IV) inhibitor such that the combination provides an effect in lowering a blood glucose level or in increasing a blood GLP-1 level in a subject over that provided by the amount of the BRS-3 agonist alone and by the amount of the DPP-IV inhibitor alone and the use of such a combination for treating or preventing obesity and diabetes and conditions related thereto and conditions ameliorated by increasing a blood GLP-1 level. The present invention also relates to the use of a G protein-coupled receptor to screen for GLP-1 secretagogues.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treatingor preventing obesity and diabetes and conditions related thereto. Thepresent invention further relates to compositions and methods forincreasing a blood GLP-1 level in a mammal. The present invention alsorelates to methods of using a G protein-coupled receptor to screen forGLP-1 secretagogues.

BACKGROUND OF THE INVENTION

The following discussion is intended to facilitate the understanding ofthe invention, but is not intended nor admitted to be prior art to theinvention.

A. Obesity and Diabetes

Obesity is the most common metabolic disease in developed nations.Despite public health education and initiatives, its prevalencecontinues to rise, with greater than 30% of adults in the United Statesbeing obese and greater than 60% of adults being overweight or obese.The World Health Organization has estimated that worldwide, over onebillion adults are overweight, with at least 300 million of them beingobese. Obesity leads to, or significantly increases the risk of, a widerange of comorbidities that includes but is not limited to hypertension,congestive cardiomyopathy, coronary heart disease, stroke, dyslipidemia,metabolic syndrome, and Type 2 diabetes [Bays, Obesity Research (2004)12:11971211] and premature death. There is an unmet medical need forsafe and effective antiobesity drugs as a therapeutic option with whichto reduce the worldwide obesity epidemic.

The incidence of Type 2 diabetes in the United States is about 7% andaccounts for as much as 10% of all health care dollars. Furthermore, theincidence of Type 2 diabetes worldwide is increasing such that Type 2diabetes is now considered to be a worldwide epidemic. Type 2 diabetesis characterized by fasting and postprandial hyperglycemia and byrelative insulin insufficiency. Hyperglycemia may cause long-termmicrovascular and macrovascular complications, such as nephropathy,neuropathy, retinopathy, and peripheral vascular disease. In addition,Type 2 diabetes is a comorbid disease that frequently compoundshyperlipidemia, atherosclerosis and hypertension. Hyperlipidemia is aprimary risk factor for cardiovascular disease due to atherosclerosis.Type 2 diabetes causes significant morbidity and mortality atconsiderable expense to patients, their families and society.

B. Glucagon-Like Peptide-1 (GLP-1)

Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from theposttranslational modification of proglucagon and secreted by gutendocrine cells. GLP-1 mediates its actions through a specific Gprotein-coupled receptor (GPCR), namely GLP-1R. GLP-1 is bestcharacterized as a hormone that regulates glucose homeostasis. GLP-1 hasbeen shown to stimulate glucose-dependent insulin secretion and toincrease pancreatic beta cell mass. GLP-1 has also been shown to reducethe rate of gastric emptying and to promote satiety. The efficacy ofGLP-1 peptide agonists in controlling blood glucose in Type 2 diabeticshas been demonstrated in several clinical studies [see, e.g., Nauck etal., Drug News Perspect (2003) 16:413-422], as has its efficacy inreducing body mass [Zander et al., Lancet (2002) 359:824-830].

GLP-1 receptor agonists are additionally useful in protecting againstmyocardial infarction and against cognitive and neurodegenerativedisorders. GLP-1 has been shown to be cardioprotective in a rat model ofmyocardial infarction [Bose et al., Diabetes (2005) 54:146-151], andGLP-1R has been shown in rodent models to be involved in learning andneuroprotection [During et al., Nat Med (2003) 9:1173-1179; and Greig etal., Ann N Y Acad Sci (2004) 1035:290-315].

Certain disorders such as Type 2 diabetes are characterized by adeficiency in GLP-1 [see, e.g., Nauck et al., Diabetes (2004) 53 Suppl3:S190-196].

Current GLP-1 peptide agonists suffer from a lack of oralbioavailability, negatively impacting patient compliance. Efforts todevelop orally bioavailable non-peptidergic, small-molecule agonists ofGLP-LR have so far been unsuccessful [Mentlein, Expert Opin InvestigDrugs (2005) 14:57-64]. An attractive alternative approach is to developan orally active composition for increasing an endogenous level of GLP-1in the blood.

C. BRS-3

Bombesin is a 14 amino acid peptide isolated from frog skin. BombesinReceptor Subtype-3 BRS-3 G protein-coupled receptor (BRS-3; e.g., humanBRS-3, GenBank® Accession No. AAA35604 and alleles thereof; e.g., mouseBRS-3, GenBank® Accession No. AY288423 and alleles thereof) exhibitsabout 50% homology to gastric-releasing peptide receptor (GRP-R) andneuromedin B receptor (NMB-R), and together they form the bombesin-likereceptor group. BRS-3 is selectively expressed in tissues includinghypothalamus and uterus. BRS-3 activation leads to increasedaccumulation of intracellular inositol 1,4,5-triphosphate (IP3),consistent with BRS-3 being coupled to Gq. In recent studies, BRS-3knockout mice developed obesity, diabetes, and hypertension[Ohki-Hamazaki et al., Nature (1997) 390:165-169].

D. Dipeptidyl Peptidase IV (DPP-IV)

Dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5) exhibits catalyticactivity against a broad range of peptide substrates that includespeptide hormones, neuropeptides, and chemokines. The incretinsglucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropicpolypeptide (GIP), which stimulate glucose-dependent insulin secretionand otherwise promote blood glucose homeostasis, are rapidly cleaved byDPP-IV at the position 2 alanine leading to inactivation of theirbiological activity. Both pharmacological and genetic attenuation ofDPP-IV activity is associated with enhanced incretin action, increasedinsulin, and lower blood glucose in vivo. Genetic attenuation of DPP-IVactivity has been shown to provide resistance to obesity and to improveinsulin sensitivity. A second-generation DPP-IV inhibitor, LAF237 (Ahrenet al., J Clin Endocrinol Metab (2004) 89:2078-2084; and Villhauer etal., J Med Chem (2003) 46:2774-2789; the disclosure of each of which isherein incorporated by reference in its entirety), is currently in phase3 clinical trials for Type 2 diabetes and additional DPP-IV inhibitorsare in clinical development.

Because the incretin hormones are not the only substrates for DPP-IV,there is concern that inhibition of the cleavage of other endogenousDPP-IV substrates may give rise to undesirable side effects [see, e.g.,Chen et al, J Biol Regul Homeost Agents (2004) 18:47-54, the disclosureof which is herein incorporated by reference in its entirety]. Ittherefore would be advantageous to identify an activity promoting bloodglucose homeostasis which is associated with substantially lowerconcentrations of DPP-IV inhibitor.

E. G Protein-Coupled Receptors

GPCRs share a common structural motif, having seven sequences of between22 to 24 hydrophobic amino acids that form seven alpha helices, each ofwhich spans the membrane (each span is identified by number, i.e.,transmembrane-1 (TM-1), transmembrane-2 (TM-2), etc.). The transmembranehelices are joined by strands of amino acids between transmembrane-2 andtransmembrane-3, transmembrane-4 and transmembrane-5, andtransmembrane-6 and transmembrane-7 on the exterior, or “extracellular”side, of the cell membrane (these are referred to as “extracellular”regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively). Thetransmembrane helices are also joined by strands of amino acids betweentransmembrane-1 and transmembrane-2, transmembrane-3 andtransmembrane-4, and transmembrane-5 and transmembrane-6 on theinterior, or “intracellular” side, of the cell membrane (these arereferred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3),respectively). The “carboxy” (“C”) terminus of the receptor lies in theintracellular space within the cell, and the “amino” (“N”) terminus ofthe receptor lies in the extracellular space outside of the cell.

Generally, when an agonist binds to a G protein-coupled receptor (oftenreferred to as “activation” of the receptor), there is a change in theconformation of the receptor that facilitates coupling between theintracellular region and an intracellular “G-protein.” It has beenreported that GPCRs are “promiscuous” with respect to G proteins, i.e.,that a GPCR can interact with more than one G protein. See, Kenakin, T.,43 Life Sciences 1095 (1988). Although other G proteins may exist,currently, Gq, Gs, Gi, Gz and Go are G proteins that have beenidentified. Ligand-activated GPCR coupling with the G-protein initiatesa signaling cascade process (referred to as “signal transduction”).Under normal conditions, signal transduction ultimately results incellular activation or cellular inhibition.

Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and Go), on theother hand, inhibit adenylyl cyclase. Adenylyl cyclase catalyzes theconversion of ATP to cAMP; thus, activated GPCRs that couple the Gsprotein are associated with increased cellular levels of cAMP. On theother hand, activated GPCRs that couple Gi (or Gz, Go) protein areassociated with decreased cellular levels of cAMP. See, generally,“Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron ToBrain (3^(rd) Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc.(1992). Thus, assays that detect cAMP can be utilized to determine if acandidate compound is, e.g., an agonist to a Gs-associated receptor(i.e., such a compound would increase the levels of cAMP). Gq and Go areassociated with activation of the enzyme phospholipase C, which in turnhydrolyzes the phospholipid PIP₂, releasing two intracellularmessengers: diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3).Increased accumulation of IP3 is associated with activation of Gq- andGo-associated receptors. See, generally, “Indirect Mechanisms ofSynaptic Transmission,” Chpt. 8, From Neuron To Brain (3^(rd) Ed.)Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992). Assays thatdetect IP3 accumulation can be utilized to determine if a candidatecompound is, e.g. an agonist to a Gq- or Go-associated receptor (i.e.,such a compound would increase the levels of IP3). Assays that detectthe level of intracellular free calcium can also be utilized todetermine if a candidate compound is, e.g., an agonist to a Gq orGo-associated receptor (i.e., such a compound would increase the levelsof intracellular free calcium).

See, e.g., Table A (“N/A”: “not applicable”).

TABLE A Effect on cAMP Production Effect on IP3 Accumulation Effect oncAMP Effect on IP3 upon Activation of GPCR upon Activation of GPCRProduction Accumulation G (i.e., constitutive activation (i.e.,constitutive activation upon contact with upon contact with protein oragonist binding) or agonist binding) an Inverse Agonist an InverseAgonist Gs Increase N/A Decrease N/A Gi Decrease N/A Increase N/A GzDecrease N/A Increase N/A Go Decrease Increase Increase Decrease Gq N/AIncrease N/A Decrease

There are also promiscuous G proteins, which appear to couple severalclasses of GPCRs to the phospholipase C pathway, such as Gα15 or Gα16[Offermanns & Simon, J Biol Chem (1995) 270:15175-80], or chimeric Gproteins designed to couple a large number of different GPCRs to thesame pathway, e.g. phospholipase C [Milligan & Rees, Trends inPharmaceutical Sciences (1999) 20:118-24]. Assays that detect the levelof intracellular free calcium can be utilized to determine if acandidate compound is, e.g., an agonist to a GPCR coupled to thephospholipase C pathway (i.e., such a compound would increase the levelsof intracellular free calcium).

Under physiological conditions, GPCRs exist in the cell membrane inequilibrium between two different conformations: an “inactive” state andan “active” state. A receptor in an inactive state is unable to link tothe intracellular signaling transduction pathway to initiate signaltransduction leading to a biological response. Changing the receptorconformation to the active state allows linkage to the transductionpathway (via the G-protein) and produces a biological response.

A receptor may be stabilized in an active state by a ligand or acompound such as a drug. Recent discoveries, including but notexclusively limited to modifications to the amino acid sequence of thereceptor, provide means other than ligands or drugs to promote andstabilize the receptor in the active state conformation. These meanseffectively stabilize the receptor in an active state by simulating theeffect of a ligand binding to the receptor. Stabilization by suchligand-independent means is termed “constitutive receptor activation.”An endogenous receptor exhibiting activity in the absence of ligand isreferred to as a constitutively active endogenous receptor.

SUMMARY OF THE INVENTION

The present invention concerns combination of an amount of a BRS-3agonist with an amount of a dipeptidyl peptidase IV (DPP-IV) inhibitorsuch that the combination provides an effect in lowering a blood glucoselevel in a subject over that provided by the amount of the BRS-3 agonistor the amount of the DPP-IV inhibitor alone and the use of such acombination for treating or preventing diabetes and conditions relatedthereto. The present invention further concerns combination of an amountof a BRS-3 agonist with an amount of a dipeptidyl peptidase IV (DPP-IV)inhibitor such that the combination provides an effect in increasing ablood GLP-1 level in a subject over that provided by the amount of theBRS-3 agonist or the amount of the DPP-IV inhibitor alone and the use ofsuch a combination for treating or preventing a condition ameliorated byincreasing a blood GLP-1 level or for increasing a blood GLP-1 level ina subject deficient in GLP-1. The present invention also relates tomethods of using BRS-3 G protein-coupled receptor to screen for GLP-1secretagogues.

In a first aspect, the present invention features a method of treatingor preventing diabetes or a condition related thereto comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising or consisting essentially of a BRS-3agonist and a DPP-IV inhibitor. In certain embodiments, the BRS-3agonist and the DPP-IV inhibitor are administered in amounts sufficientto lower a blood glucose level in the subject. In certain embodiments,the blood glucose level is an elevated blood glucose level.

The present invention additionally features a method of reducing bodymass comprising administering to a subject in need thereof atherapeutically effective amount of a composition comprising orconsisting essentially of a BRS-3 agonist and a DPP-IV inhibitor. Incertain embodiments, the BRS-3 agonist and the DPP-IV inhibitor areadministered in amounts sufficient to increase a blood GLP-1 level inthe subject. In certain embodiments, the subject in need thereof has aBMI of 27 or greater. In certain embodiments, the subject in needthereof is overweight. In certain embodiments, the subject in needthereof is obese.

The present invention additionally features a method of treating orpreventing obesity or a condition related thereto comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising or consisting essentially of a BRS-3agonist and a DPP-IV inhibitor. In certain embodiments, the BRS-3agonist and the DPP-IV inhibitor are administered in amounts sufficientto increase a blood GLP-1 level in the subject.

The present invention additionally features a method of treating orpreventing a condition ameliorated by increasing a blood GLP-1 levelcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a composition comprising or consisting essentiallyof a BRS-3 agonist and a DPP-IV inhibitor. In certain embodiments, theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to increase a blood GLP-1 level in the subject.

The present invention additionally features a method of increasing ablood GLP-1 level comprising administering to a subject deficient inGLP-1 a therapeutically effective amount of a composition comprising orconsisting essentially of a BRS-3 agonist and a DPP-IV inhibitor. Incertain embodiments, the BRS-3 agonist and the DPP-IV inhibitor areadministered in amounts sufficient to increase a blood GLP-1 level inthe subject.

In certain embodiments, diabetes is Type 2 diabetes.

In certain embodiments, the condition related to diabetes is selectedfrom the group consisting of hyperglycemia, impaired glucose tolerance,insulin resistance, pancreatic beta-cell insufficiency, enteroendocrinecell insufficiency, glucosuria, metabolic acidosis, cataracts, diabeticnephropathy, diabetic neuropathy, diabetic retinopathy, diabeticcoronary artery disease, diabetic cerebrovascular disease, diabeticperipheral vascular disease, metabolic syndrome, hyperlipidemia,atherosclerosis, stroke, hypertension, and obesity.

In certain embodiments, the condition related to obesity is selectedfrom the group consisting of hypertension, congestive cardiomyopathy,varicosities, pulmonary embolism, coronary heart disease, stroke,idiopathic intracranial hypertension, meralgia parethetica, dyspnea,obstructive sleep apnea, hyperventilation syndrome, Pickwickiansyndrome, asthma, immobility, degenerative osteoarthritis, low backpain, striae distensae or “stretch marks,” venous stasis of the lowerextremities, lymphedema, cellulitis, intertrigo, carbuncles, acanthosisnigricans, skin tags, gastro-esophageal reflux disorder, nonalcoholicfatty liver/steatohepatitis, cholelithiasis, hernias, colon cancer,stress incontinence, obesity-related glomerulopathy, breast and uterinecancer, depression and low self-esteem, impaired quality of life,metabolic syndrome, Type 2 diabetes, dyslipidemia, hyperandrogenemia inwomen, polycystic ovarian syndrome, dysmenorrhea, infertility, pregnancycomplications, and male hypogonadism. In certain embodiments, thecondition related to obesity is selected from the group consisting ofhypertension, congestive cardiomyopathy, coronary heart disease, stroke,dyslipidemia, metabolic syndrome, and Type 2 diabetes.

In certain embodiments, the condition ameliorated by increasing a bloodGLP-1 level is selected from the group consisting of diabetes, acondition related to diabetes, myocardial infarction, learningimpairment, memory impairment, and a neurodegenerative disorder.

In certain embodiments, the condition ameliorated by increasing a bloodGLP-1 level is a neurodegenerative disorder selected from the groupconsisting of excitotoxic brain damage caused by severe epilepticseizures, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, prion-associated disease, stroke, motor-neuron disease,learning or memory impairment, traumatic brain injury, spinal cordinjury, and peripheral neuropathy.

In certain embodiments, the subject is a human.

In a second aspect, the present invention features a compositioncomprising or consisting essentially of a BRS-3 agonist and a DPP-IVinhibitor. In certain embodiments, the present invention relates to adosage form of the composition wherein the BRS-3 agonist and the DPP-IVinhibitor are in amounts sufficient to lower a blood glucose level in asubject. In certain embodiments, the blood glucose level is an elevatedblood glucose level. In certain embodiments, the present inventionrelates to a dosage form of the composition wherein the BRS-3 agonistand the DPP-IV inhibitor are in amounts sufficient to increase a bloodGLP-1 level in a subject.

In certain embodiments, the subject is a human.

In a third aspect, the present invention features a compositioncomprising or consisting essentially of a BRS-3 agonist and a DPP-IVinhibitor for use in a method of treatment of the human or animal bodyby therapy. In certain embodiments, the present invention relates to adosage form of the composition wherein the BRS-3 agonist and the DPP-IVinhibitor are in amounts sufficient to lower a blood glucose level in asubject. In certain embodiments, the blood glucose level is an elevatedblood glucose level. In certain embodiments, the present inventionrelates to a dosage form of the composition wherein the BRS-3 agonistand the DPP-IV inhibitor are in amounts sufficient to increase a bloodGLP-1 level in a subject.

The present invention additionally features a composition comprising orconsisting essentially of a BRS-3 agonist and a DPP-IV inhibitor for usein a method of treatment or prevention of diabetes or a conditionrelated thereto of the human or animal body by therapy. In certainembodiments, the present invention relates to a dosage form of thecomposition wherein the BRS-3 agonist and the DPP-IV inhibitor are inamounts sufficient to lower a blood glucose level in a subject. Incertain embodiments, the blood glucose level is an elevated bloodglucose level.

The present invention additionally features a composition comprising orconsisting essentially of a BRS-3 agonist and a DPP-IV inhibitor for usein a method of reducing body mass of the human or animal body bytherapy. In certain embodiments, the present invention relates to adosage form of the composition wherein the BRS-3 agonist and the DPP-IVinhibitor are in amounts sufficient to increase a blood GLP-1 level in asubject.

The present invention additionally features a composition comprising orconsisting essentially of a BRS-3 agonist and a DPP-IV inhibitor for usein a method of treatment or prevention of obesity or a condition relatedthereto of the human or animal body by therapy. In certain embodiments,the present invention relates to a dosage form of the compositionwherein the BRS-3 agonist and the DPP-IV inhibitor are in amountssufficient to increase a blood GLP-1 level in a subject.

The present invention additionally features a composition comprising orconsisting essentially of a BRS-3 agonist and a DPP-IV inhibitor for usein a method of treatment or prevention of a condition ameliorated byincreasing a blood GLP-1 level of the human or animal body by therapy.In certain embodiments, the present invention relates to a dosage formof the composition wherein the BRS-3 agonist and the DPP-IV inhibitorare in amounts sufficient to increase a blood GLP-1 level in a subject.

The present invention additionally features a composition comprising orconsisting essentially of a BRS-3 agonist and a DPP-IV inhibitor for usein a method of treatment or prevention of a deficiency of GLP-1 of thehuman or animal body by therapy. In certain embodiments, the presentinvention relates to a dosage form of the composition wherein the BRS-3agonist and the DPP-IV inhibitor are in amounts sufficient to increase ablood GLP-1 level in a subject.

In certain embodiments, the subject is a human.

In a fourth aspect, the present invention features a method of preparinga pharmaceutical composition, said method comprising or consistingessentially of admixing a BRS-3 agonist and a DPP-IV inhibitor, togetherwith at least one pharmaceutically acceptable carrier. In certainembodiments, the method further comprises the step of preparing a dosageform of the pharmaceutical composition wherein the BRS-3 agonist and theDPP-IV inhibitor are in amounts sufficient to lower a blood glucoselevel in a subject. In certain embodiments, the blood glucose level isan elevated blood glucose level. In certain embodiments, the methodfurther comprises the step of preparing a dosage form of thepharmaceutical composition wherein the BRS-3 agonist and the DPP-IVinhibitor are in amounts sufficient to increase a blood GLP-1 level in asubject.

In certain embodiments, the subject is a human.

In a fifth aspect, the present invention features a pharmaceuticalcomposition comprising or consisting essentially of a BRS-3 agonist anda DPP-IV inhibitor, together with at least one pharmaceuticallyacceptable carrier. In certain embodiments, the present inventionrelates to a dosage form of the pharmaceutical composition wherein theBRS-3 agonist and the DPP-IV inhibitor are in amounts sufficient tolower a blood glucose level in a subject. In certain embodiments, theblood glucose level is an elevated blood glucose level. In certainembodiments, the present invention relates to a dosage form of thepharmaceutical composition wherein the BRS-3 agonist and the DPP-IVinhibitor are in amounts sufficient to increase a blood GLP-1 level in asubject.

In certain embodiments, the subject is a human.

In a sixth aspect, the present invention features a method of treatingor preventing diabetes or a condition related thereto comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition in accordance with the fifthaspect. In certain embodiments, the BRS-3 agonist and the DPP-IVinhibitor are administered in amounts sufficient to lower a bloodglucose level in the subject. In certain embodiments, the blood glucoselevel is an elevated blood glucose level. In certain embodiments, theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to increase a blood GLP-1 level in the subject.

The present invention additionally features a method of reducing bodymass comprising administering to a subject in need thereof atherapeutically effective amount of a pharmaceutical composition inaccordance with the fifth aspect. In certain embodiments, the BRS-3agonist and the DPP-IV inhibitor are administered in amounts sufficientto increase a blood GLP-1 level in the subject. In certain embodiments,the subject in need thereof has a BMI of 27 or greater. In certainembodiments, the subject in need thereof is overweight. In certainembodiments, the subject in need thereof is obese.

The present invention additionally features a method of treating orpreventing obesity or a condition related thereto comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition in accordance with the fifthaspect. In certain embodiments, the BRS-3 agonist and the DPP-IVinhibitor are administered in amounts sufficient to increase a bloodGLP-1 level in the subject.

The present invention additionally features a method of treating orpreventing a condition ameliorated by increasing a blood GLP-1 levelcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a pharmaceutical composition in accordance with thefifth aspect. In certain embodiments, the BRS-3 agonist and the DPP-IVinhibitor are administered in amounts sufficient to increase a bloodGLP-1 level in the subject.

The present invention additionally features a method of increasing ablood GLP-1 level comprising administering to a subject deficient inGLP-1 a therapeutically effective amount of a pharmaceutical compositionin accordance with the fifth aspect. In certain embodiments, the BRS-3agonist and the DPP-IV inhibitor are administered in amounts sufficientto increase a blood GLP-1 level in the subject.

In certain embodiments, the subject is a human.

In a seventh aspect, the present invention features use of a compositioncomprising or consisting essentially of a BRS-3 agonist and a DPP-IVinhibitor for the manufacture of a medicament for the treatment orprevention of diabetes or a condition related thereto. In certainembodiments, the present invention relates to a dosage form of themedicament wherein the BRS-3 agonist and the DPP-IV inhibitor are inamounts sufficient to lower a blood glucose level in a subject. Incertain embodiments, the blood glucose level is an elevated bloodglucose level. In certain embodiments, the present invention relates toa dosage form of the medicament wherein the BRS-3 agonist and the DPP-IVinhibitor are in amounts sufficient to increase a blood GLP-1 level in asubject.

The present invention additionally features use of a compositioncomprising or consisting essentially of a BRS-3 agonist and a DPP-IVinhibitor for the manufacture of a medicament for reducing body mass. Incertain embodiments, the present invention relates to a dosage form ofthe medicament wherein the BRS-3 agonist and the DPP-IV inhibitor are inamounts sufficient to increase a blood GLP-1 level in a subject.

The present invention additionally features use of a compositioncomprising or consisting essentially of a BRS-3 agonist and a DPP-IVinhibitor for the manufacture of a medicament for the treatment orprevention of obesity or a condition related thereto. In certainembodiments, the present invention relates to a dosage form of themedicament wherein the BRS-3 agonist and the DPP-IV inhibitor are inamounts sufficient to increase a blood GLP-1 level in a subject.

The present invention additionally features use of a compositioncomprising or consisting essentially of a BRS-3 agonist and a DPP-IVinhibitor for the manufacture of a medicament for the treatment orprevention of a condition ameliorated by increasing a blood GLP-1 level.In certain embodiments, the present invention relates to a dosage formof the medicament wherein the BRS-3 agonist and the DPP-IV inhibitor arein amounts sufficient to increase a blood GLP-1 level in a subject.

The present invention additionally features use of a compositioncomprising or consisting essentially of a BRS-3 agonist and a DPP-IVinhibitor for the manufacture of a medicament for the treatment orprevention of a deficiency of GLP-1. In certain embodiments, the presentinvention relates to a dosage form of the medicament wherein the BRS-3agonist and the DPP-IV inhibitor are in amounts sufficient to increase ablood GLP-1 level in a subject.

In certain embodiments, the subject is a human.

In an eighth aspect, the invention features a method for identifyingGLP-1 secretagogues or compounds useful for reducing body mass orcompounds useful for treating or preventing a condition ameliorated byincreasing a blood GLP-1 level, comprising the steps of:

-   -   (a) contacting a test compound with a host cell or with membrane        of a host cell that expresses a G protein-coupled receptor,        wherein the G protein-coupled receptor comprises an amino acid        sequence selected from the group consisting of:        -   (i) amino acids 1-335 of SEQ ID NO:2;        -   (ii) amino acids 1-399 of SEQ ID NO:2;        -   (iii) amino acids 2-335 of SEQ ID NO:2;        -   (iv) amino acids 2-399 of SEQ ID NO:2;        -   (v) amino acids 2-335 of SEQ ID NO:2, with the proviso that            the receptor does not comprise the methionine residue at            amino acid position 1 of SEQ ID NO:2;        -   (vi) amino acids 2-399 of SEQ ID NO:2, with the proviso that            the receptor does not comprise the methionine residue at            amino acid position 1 of SEQ ID NO:2;        -   (vii) the amino acid sequence of a G protein-coupled            receptor encoded by a polynucleotide comprising a nucleotide            sequence, said nucleotide sequence being the sequence            obtainable by a process comprising performing polymerase            chain reaction (PCR) on a human DNA sample using specific            primers SEQ ID NO:3 and SEQ ID NO:4;        -   (viii) the amino acid sequence of a G protein-coupled            receptor encoded by a polynucleotide comprising a nucleotide            sequence, said nucleotide sequence hybridizing under            stringent conditions to the complement of SEQ ID NO:1; and        -   (ix) a biologically active fragment of any one of (i) to            (viii); and    -   (b) determining the ability of the test compound to stimulate        functionality of the receptor;        wherein the ability of the test compound to stimulate        functionality of the receptor is indicative of the test compound        being a GLP-1 secretagogue or a compound useful for reducing        body mass or a compound useful for preventing or treating a        condition ameliorated by increasing a blood GLP-1 level.

In certain embodiments, the method is a method for identifying GLP-1secretagogues.

In certain embodiments, the method is a method for identifying compoundsuseful for reducing body mass.

In certain embodiments, the method is a method for identifying compoundsuseful for treating or preventing a condition ameliorated by increasinga blood GLP-1 level.

The invention additionally features a method for identifying GLP-1secretagogues or compounds useful for reducing body mass or compoundsuseful for treating or preventing a condition ameliorated by increasinga blood GLP-1 level, comprising steps (a) and (b) of this eighth aspect,and further comprising:

-   -   (c) contacting a compound which stimulates functionality of the        receptor in step (b) in vitro with a mammalian enteroendocrine        cell; and    -   (d) determining whether the compound stimulates GLP-1 secretion        from the mammalian enteroendocrine cell;

wherein the ability of the test compound to stimulate GLP-1 secretionfrom the mammalian enteroendocrine cell is indicative of the testcompound being a GLP-1 secretagogue or a compound useful for reducingbody mass or a compound useful for treating or preventing a conditionameliorated by increasing a blood GLP-1 level. In certain embodiments,the mammalian enteroendocrine cell is GLUTag enteroendocrine L-cellline.

The invention additionally features a method for identifyng GLP-1secretagogues or compounds useful for reducing body mass or compoundsuseful for treating or preventing a condition ameliorated by increasinga blood GLP-1 level, comprising steps (a) and (b) of this eighth aspect,and further comprising:

-   -   (c) administering a compound which stimulates functionality of        the receptor in step (b) to a mammal; and    -   (d) determining whether the compound increases a blood GLP-1        level in the mammal;

wherein the ability of the test compound to increase a blood GLP-1 levelin the mammal is indicative of the test compound being a GLP-1secretagogue or a compound useful for reducing body mass or a compounduseful for treating or preventing a condition ameliorated by increasinga blood GLP-1 level. In certain embodiments, the mammal is a non-humanmammal.

In certain embodiments, the identified GLP-1 secretagogue or theidentified compound useful for reducing body mass or the identifiedcompound useful for treating or preventing a condition ameliorated byincreasing a blood GLP-1 level is an agonist of the receptor. In someembodiments, the agonist is a partial agonist.

In certain embodiments, receptor is coupled to a G protein. In certainembodiments, the G protein is Gq.

In certain embodiments, the process is RT-PCR (reversetranscription-polymerase chain reaction). RT-PCR techniques are wellknown to the skilled artisan.

In certain embodiments, the human DNA sample is human cDNA. In certainembodiments, the cDNA is from a human tissue that expresses BRS-3. Insome embodiments, the human tissue that expresses BRS-3 is hypothalamusor uterus.

In certain embodiments, stringent hybridization conditions comprisehybridization at 42° C. in a solution comprising 50% formamide, 5×SSC(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing at 65° C. in a solutioncomprising 0.1×SSC. Hybridization techniques are well known to theskilled artisan.

In certain embodiments, the G protein-coupled receptor encoded by apolynucleotide comprising a nucleotide sequence, said nucleotidesequence hybridizing under stringent conditions to the complement of SEQID NO:1, exhibits a biological activity selected from the groupconsisting of increasing a level of intracellular IP3 and binding aknown ligand of BRS-3.

In certain embodiments, the encoded G protein-coupled receptor increasesa level of intracellular IP3 and binds a known ligand of BRS-3.

In some embodiments, the G protein-coupled receptor is part of a fusionprotein comprising a G protein. Techniques for mailing a GPCR:G fusionconstruct are well known to the skilled artisan (see, e.g.,International Application WO 02/42461).

In some embodiments, the G protein-coupled receptor is recombinant.

In certain embodiments, the host cell comprises an expression vector,said expression vector comprising a polynucleotide encoding the Gprotein-coupled receptor. In some embodiments, the expression vector ispCMV. This vector was deposited with the American Type CultureCollection (ATCC) on Oct. 13, 1998 (10801 University Blvd., Manassas,Va. 20110-2209 USA) under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganisms for thePurpose of patent Procedure. The DNA was tested by the ATCC anddetermined to be viable. The ATCC has assigned the following depositnumber to pCMV: ATCC #203351. Other suitable expression vectors will bereadily apparent to those of ordinary skill in the art, and a widevariety of expression vectors are commercially available (e.g., fromClontech, Palo Alto, Calif.; Stratagene, La Jolla, Calif.; andInvitrogen, Carlsbad, Calif.).

Suitable host cells of the invention include any eukaryotic cell capableof expressing a G protein-coupled receptor of the invention. Theeukaryotic cell can be an animal cell (e.g., an insect, mammal, fish,amphibian, bird or reptile cell), a plant cell (for example a maize orArabidopsis cell), or a fungal cell (for example a S. cerevisiae cell).In some embodiments, the host cell is mammalian. Exemplary mammalianhost cells include but are not limited to: monkey kidney cells (COScells), monkey kidney CVI cells transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney cells (HEK-293 [“293”], Graham et al. J.Gen Virol. 36:59 (1977)); human embryonic kidney cells (HEK-293T[“293T”], originally referred to as 293tsA1609neo, DuBridge et al. MolCell Biol 7:379-387 (1987)); baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary-cells (CHO, Urlaub and Chasin, Proc. Natl.Acad. Sci. (USA) 77:4216, (1980); Syrian golden hamster cells MCB3901(ATCC CRL-9595); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); african greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (IELA, ATCC CCL 2); canine kidney cells (OMCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL 51); TR1 cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); NIW3T3 cells (ATCC CRL-1658); and mouse L cells(ATCC CCL-1). In some embodiments, the mammalian host cell is selectedfrom the group consisting of 293, 293T, CHO, MCB3901, and COS-7. Incertain embodiments, melanophore cells are used. In some embodiments,the host cell is an insect cell (for example a Spodoptera frugiperdainsect Sf9 cell (ATCC CRL-1711)). In some embodiments, the host cell isa fungal cell (for example a S. cerevisiae cell). In some embodiments,the host cell is an enteroendocrine cell. In some embodiments, theenteroendocrine cell is GLUTag enteroendocrine L-cell line. Othersuitable host cells will be readily apparent to those of ordinary skillin the art, and a wide variety of cell lines are available from theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209.

In certain embodiments, said determining is consistent with the Gprotein-coupled receptor being coupled to Gq.

In some embodiments, said determining is consistent with the Gprotein-coupled receptor being coupled through a promiscuous G protein,such as Gα15 or Gα16, to the phopholipase C pathway. Promiscuous Gproteins are well known to the skilled artisan [see, e.g., Offermanns etal., J Biol Chem (1995) 270:15175-15180]. In some embodiments, saiddetermining is consistent with the G protein-coupled receptor beingcoupled through a chimeric G protein, e.g. to the phospholipase Cpathway. Chimeric G proteins are well known to the skilled artisan [see,e.g., Milligan et al., Trends in Pharmaceutical Sciences (1999)20:118-124; and WO 02/42461].

In some embodiments, said determining is through the measurement of alevel of a second messenger.

In some embodiments, said determining is through the measurement of alevel of a second messenger selected from the group consisting of cyclicAMP (cAMP), cyclic GMP (cGMP), inositol 1,4,5-triphosphate (IP3),diacylglycerol (DAG), MAP kinase activity, MAPK/ERK kinase kinase-1(MEKK1) activity, and Ca2+. In some preferred embodiments, the secondmessenger is IP3. In certain preferred embodiments, a level ofintracellular IP3 is increased. In some preferred embodiments, thesecond messenger is Ca2+. In certain preferred embodiments, a level ofintracellular Ca2+ is increased. In some embodiments, said Ca2+measurement is carried out by FLIPR.

In certain embodiments, said determining is carried out with membranecomprising the G protein-coupled receptor.

In certain embodiments, said determining is through the use of amelanophore assay. In some preferred embodiments, a level of pigmentdispersion is increased.

In some embodiments, said determining is through the measurement of anactivity mediated by elevation of a level of intracellular IP3. In someembodiments, said activity is stimulation of GLP-1 secretion.

In some embodiments, said determining is through AP1-reporter assay. Insome embodiments, said determining is through SRF-reporter assay. Insome embodiments, said reporter is luciferase. In some embodiments, saidreporter is β-galactosidase. In certain embodiments, a level ofluciferase activity or β-galactosidase activity is increased.

In some embodiments, said determining is through the measurement ofGTPγS binding to membrane comprising the G protein-coupled receptor. Insome preferred embodiments, said GTPγS is labeled with [³⁵S]. In somepreferred embodiments, said GTPγS binding to membrane comprising theGPCR is increased.

In some embodiments, the candidate compound is a small molecule. In someembodiments, the candidate compound is a small molecule, with theproviso that the small molecule is not a polypeptide. In someembodiments, the candidate compound is a small molecule, with theproviso that the small molecule is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the candidate compound is a smallmolecule, with the proviso that the small molecule is not a lipid. Insome embodiments, the candidate compound is a small molecule, with theproviso that the small molecule is not a polypeptide or a lipid. In someembodiments, the candidate compound is a polypeptide. In someembodiments, the candidate compound is a polypeptide, with the provisothat the polypeptide is not an antibody or an antigen-binding fragmentthereof. In some embodiments, the candidate compound is a lipid. In someembodiments, the candidate compound is not an antibody or anantigen-binding fragment thereof. In some embodiments, the candidatecompound is an antibody or an antigen-binding fragment thereof.

In some embodiments, the method further comprises synthesizing the GLP-1secretagogue or the compound useful for treating or preventing acondition ameliorated by increasing a blood GLP-1 level.

In some embodiments, the method further comprises: optionally,determining the structure of the GLP-1 secretagogue or the compounduseful for reducing body mass or the compound useful for treating orpreventing a condition ameliorated by increasing a blood GLP-1 level;and providing the GLP-1 secretagogue or the compound useful for reducingbody mass or the compound useful for treating or preventing a conditionameliorated by increasing a blood GLP-1 level or providing the name orstructure of the GLP-1 secretagogue or the compound useful for reducingbody mass or the compound useful for treating or preventing a conditionameliorated by increasing a blood GLP-1 level.

In some embodiments, said method further comprises: optionally,determining the structure of the GLP-1 secretagogue or the compounduseful for reducing body mass or the compound useful for treating orpreventing a condition ameliorated by increasing a blood GLP-1 level;optionally, providing the name or structure of the GLP-1 secretagogue orthe compound useful for reducing body mass or the compound useful fortreating or preventing a condition ameliorated by increasing a bloodGLP-1 level; and producing or synthesizing the GLP-1 secretagogue or thecompound useful for reducing body mass or the compound useful fortreating or preventing a condition ameliorated by increasing a bloodGLP-1 level.

In a ninth aspect, the invention features a method for identifying GLP-1secretagogues or compounds useful for reducing body mass or compoundsuseful for treating or preventing a condition ameliorated by increasinga blood GLP-1 level, comprising the steps of:

-   -   (a) contacting a G protein-coupled receptor with an optionally        labeled known ligand to the receptor in the presence or absence        of a test compound, wherein the G protein-coupled receptor        comprises an amino acid sequence selected from the group        consisting of:        -   (i) amino acids 1-335 of SEQ ID NO:2;        -   (ii) amino acids 1-399 of SEQ ID NO:2;        -   (iii) amino acids 2-335 of SEQ ID NO:2;        -   (iv) amino acids 2-399 of SEQ ID NO:2;        -   (v) amino acids 2-335 of SEQ ID NO:2, with the proviso that            the receptor does not comprise the methionine residue at            amino acid position 1 of SEQ ID NO:2;        -   (vi) amino acids 2-399 of SEQ ID NO:2, with the proviso that            the receptor does not comprise the methionine residue at            amino acid position 1 of SEQ ID NO:2;        -   (vii) the amino acid sequence of a G protein-coupled            receptor encoded by a polynucleotide comprising a nucleotide            sequence, said nucleotide sequence being the sequence            obtainable by a process comprising performing polymerase            chain reaction (PCR) on a human DNA sample using specific            primers SEQ ID NO:3 and SEQ ID NO:4;        -   (viii) the amino acid sequence of a G protein-coupled            receptor encoded by a polynucleotide comprising a nucleotide            sequence, said nucleotide sequence hybridizing under            stringent conditions to the complement of SEQ ID NO:1; and        -   (ix) a biologically active fragment of any one of (i) to            (viii); and    -   (b) detecting the complex between said known ligand and said        receptor, and    -   (c) determining whether less of said complex is formed in the        presence of the test compound than in the absence of the test        compound;        wherein said determination is indicative of the test compound        being a GLP-1 secretagogue or a compound useful for reducing        body mass or a compound useful for preventing or treating a        condition ameliorated by increasing a blood GLP-1 level.

In certain embodiments, the method is a method for identifying GLP-1secretagogues.

In certain embodiments, the method is a method for identifying compoundsuseful for reducing body mass.

In certain embodiments, the method is a method for identifying compoundsuseful for treating or preventing a condition ameliorated by increasinga blood GLP-1 level.

In certain embodiments, the known ligand is selected from the groupconsisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16,Compound 17, Compound 18, Compound 19, Compound 20, Compound 21,Compound 22, Compound 23, Compound 24, Compound 25, Compound 26,Compound 27, Compound 28, Compound 29, Compound 30, Compound 31,Compound 32, Compound 33, and Compound 34.

In certain embodiments, the optionally labeled known ligand is a labeledknown ligand. In certain embodiments, the labeled known ligand is aradiolabeled known ligand. Techniques for radiolabeling a compound, suchas for labeling a known ligand of a G protein-coupled receptor of theinvention, are well known to the skilled artisan. See, e.g.,International Application WO 04/065380.

Techniques for detecting the complex between a G protein-coupledreceptor and a compound known to be a ligand of the G protein-coupledreceptor are well known to the skilled artisan. See, e.g., InternationalApplication WO 04/065380.

The invention additionally features a method for identifying GLP-1secretagogues or compounds useful for reducing body mass or compoundsuseful for treating or preventing a condition ameliorated by increasinga blood GLP-1 level, comprising steps (a) to (c) of this ninth aspect,and further comprising:

-   -   (d) contacting a compound in the presence of which less of said        complex is formed in step (c) in vitro with a mammalian        enteroendocrine cell; and    -   (e) determining whether the compound stimulates GLP-1 secretion        from the mammalian enteroendocrine cell;

wherein the ability of the test compound to stimulate GLP-1 secretionfrom the mammalian enteroendocrine cell is indicative of the testcompound being a GLP-1 secretagogue or a compound useful for reducingbody mass or a compound useful for treating or preventing a conditionameliorated by increasing a blood GLP-1 level. In certain embodiments,the mammalian enteroendocrine cell is GLUTag enteroendocrine L-cellline.

The invention additionally features a method for identifying GLP-1secretagogues or compounds useful for reducing body mass or compoundsuseful for treating or preventing a condition ameliorated by increasinga blood GLP-1 level, comprising steps (a) to (c) of this ninth aspect,and further comprising:

-   -   (d) administering a compound in the presence of which less of        said complex is formed in step (c) to a mammal; and    -   (e) determining whether the compound increases a blood GLP-1        level in the mammal;        wherein the ability of the test compound to increase a blood        GLP-1 level in the mammal is indicative of the test compound        being a GLP-1 secretagogue or a compound useful for reducing        body mass or a compound useful for treating or preventing a        condition ameliorated by increasing a blood GLP-1 level. In        certain embodiments, the mammal is a non-human mammal.

In certain embodiments, the receptor is recombinant.

This application claims the benefit of priority from the followingprovisional application, filed via U.S. Express mail with the UnitedStates Patent and Trademark Office on the indicated date: U.S.Provisional No. 60/675,730, filed Apr. 27, 2005. The disclosure of theforegoing application is herein incorporated by reference in itsentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated in connection with the figures appendedhereto in which:

FIG. 1 shows increase in intracellular IP3 accumulation by BRS-3. SeeExample 9.

FIG. 2 shows expression of BRS-3 in GLUTag cells. See Example 10.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is concerned with the combination of certaincompounds, or pharmaceutically acceptable salts thereof, for thetreatment or prevention of diabetes and conditions related thereto. Thepresent invention is additionally concerned with the combination ofcertain compounds, or pharmaceutically acceptable salts thereof, forreducing body mass. The present invention is additionally concerned withthe combination of certain compounds, or pharmaceutically acceptablesalts thereof, for the treatment or prevention of obesity and conditionsrelated thereto. The present invention is additionally concerned withthe combination of certain compounds, or pharmaceutically acceptablesalts thereof, for the treatment or prevention of a conditionameliorated by increasing a blood GLP-1 level. The present invention isadditionally concerned with methods of using a G protein-coupledreceptor of the invention to screen test compounds as GLP-1secretagogues. Applicant has discovered that BRS-3 is expressed bymammalian GLP-1 producing enteroendocrine L-cell. BRS-3 is a GLP-1secretagogue receptor. Agonists of BRS-3 are GLP-1 secretagogues.

By the use of a combination of a BRS-3 agonist and a DPP-IV inhibitor inaccordance with the present invention, it is possible to treat orprevent diabetes and conditions related thereto with a dose of a DPP-IVinhibitor substantially lower than that currently contemplated for usein therapy for diabetes and conditions related thereto, thereby reducingthe likelihood of unwanted side-effects associated with inhibition ofDPP-IV activity. By the use of a combination of a BRS-3 agonist and aDPP-IV inhibitor in accordance with the present invention, it ispossible to reduce body mass with a dose of a DPP-IV inhibitorsubstantially lower than that currently contemplated for reducing bodymass, thereby reducing the likelihood of unwanted side-effectsassociated with inhibition of DPP-IV activity. By the use of acombination of a BRS-3 agonist and a DPP-IV inhibitor in accordance withthe present invention, it is possible to treat or prevent obesity andconditions related thereto with a dose of a DPP-rV inhibitorsubstantially lower than that currently contemplated for use in therapyfor obesity and conditions related thereto, thereby reducing thelikelihood of unwanted side-effects associated with inhibition of DPP-IVactivity. By the use of a combination of a BRS-3 agonist and a DPP-IVinhibitor in accordance with the present invention, it is possible totreat or prevent a condition ameliorated by increasing a blood GLP-1level with a dose of a DPP-IV inhibitor substantially lower than thatcurrently contemplated for use in therapy for said condition, therebyreducing the likelihood of unwanted side-effects associated withinhibition of DPP-IV activity. Furthermore, by the use of a combinationof a BRS-3 agonist and a DPP-IV inhibitor in accordance with the presentinvention, it is possible to treat or prevent diabetes or obesity andconditions related thereto with a dose of a BRS-3 agonist substantiallylower than that currently contemplated for use in therapy for diabetesor obesity and conditions related thereto, thereby reducing thelikelihood of unwanted side-effects should any be found to be associatedwith activation of BRS-3 receptor. The present invention provides a new,unexpected and advantageous approach to lowering a blood glucose levelin a subject. The present invention additionally provides a new,unexpected and advantageous approach to increasing a blood GLP-1 levelin a subject. The present invention additionally provides a new,unexpected and advantageous approach to screening a test compound as aGLP-1 secretagogue.

The term “ligand”, as used herein, shall mean a molecule thatspecifically binds to a GPCR. A ligand may be, for example, apolypeptide, a lipid, a small molecule, an antibody. An endogenousligand is a ligand that is an endogenous, natural ligand for a nativeGPCR. A ligand may be a GPCR “antagonist”, “agonist”, “partial agonist”,or “inverse agonist”, or the like.

The term “agonist”, as used herein, shall mean an agent (e.g., ligand,candidate compound) that by virtue of binding to a GPCR activates theGPCR so as to elicit an intracellular response mediated by the GPCR.

The term “partial agonist”, as used herein, shall mean an agent (e.g.,ligand, candidate compound) that by virtue of binding to a GPCRactivates the GPCR so as to elicit an intracellular response mediated bythe GPCR, albeit to a lesser extent or degree than does a full agonist.

The term “antagonist” shall mean an agent (e.g., ligand, candidatecompound) that binds, and preferably binds competitively, to a GPCR atabout the same site as an agonist or partial agonist but which does notactivate an intracellular response initiated by the active form of theGPCR, and can thereby inhibit the intracellular response by agonist orpartial agonist. An anatagonist typically does not diminish the baselineintracellular response in the absence of an agonist or partial agonist.

The term “inverse agonist” shall mean an agent (e.g., ligand, candidatecompound) which binds to a GPCR and which inhibits the baselineintracellular response initiated by the active form of the receptorbelow the normal base level activity which is observed in the absence ofan agonist or partial agonist.

The term “BRS-3 agonist,” as used herein, refers to a compound thatbinds to BRS-3 receptor and acts as an agonist.

The term “selective BRS-3 agonist,” as used herein, refers to a BRS-3agonist having selectivity for BRS-3 receptor over one or more closelyrelated receptors, such as gastric-releasing peptide receptor (GRP-R) orneuromedin B receptor (NMB-R).

The term “DPP-IV inhibitor,” as used herein, refers to a compound thatbinds to DPP-IV and inhibits DPP-IV dipeptidyl peptidase activity.

The term “selective DPP-IV inhibitor,” as used herein, refers to aDPP-IV inhibitor having selectivity for DPP-IV over closely relatedpeptidases, such as one or more of post-proline-cleaving enzyme (PPCE),dipeptidyl peptidase II (DPP-II), dipeptidyl peptidase 8 (DPP-8), anddipeptidyl peptidase 9 (DPP-9).

The term “blood glucose level” or “blood GLP-1 level” shall mean bloodglucose concentration or blood GLP-1 concentration, respectively. Incertain embodiments, blood GLP-1 level is a level in blood ofbiologically active GLP-1, wherein GLP-1 having agonist activity atGLP-1R is biologically active. In certain embodiments, a blood glucoselevel or blood GLP-1 level is a plasma glucose level or a plasma GLP-1level.

The term “elevated blood glucose level” shall mean an elevated bloodglucose level such as that found in a subject demonstrating clinicallyinappropriate basal and postprandial hyperglycemia or such as that foundin a subject in oral glucose tolerance test (oGTT).

The term “subject,” as used herein, shall refer to a mammal, includingbut not limited to a mouse, a rat, a rabbit, a pig, a dog, a cat, anon-human primate and a human, more preferably to a mouse or rat, mostpreferably to a human.

The term “in need of prevention or treatment” as used herein refers to ajudgement made by a caregiver (e.g. physician, nurse, nurse practitionerin the case of humans; veterinarian in the case of non-human mammals)that a subject requires or will benefit from treatment.

The term “therapeutically effective amount” or “therapeuticallyeffective dose” is intended to mean that amount of drug that will elicitthe desired biological or medical response. In certain embodiments, atherapeutically effective amount is that amount of drug which willcreate an AUC inhibition above 30% in mouse oGTT assay.

The term “therapeutically ineffective amount” or “therapeuticallyineffective dose” is intended to mean an amount of drug less than thetherapeutically effective amount of the drug. In certain embodiments, atherapeutically ineffective amount is an amount of drug which willcreate an AUC inhibition less than or equal to 30% in mouse oGTT assay.

The term “amount that is effective to prevent” refers to that amount ofdrug that will prevent or reduce the risk of occurrence of thebiological or medical event that is sought to be prevented. In manyinstances, the amount that is effective to prevent is the same as thetherapeutically effective amount.

The term “composition” shall mean a material comprising at least onecomponent.

The term “active ingredient” shall mean any component that providespharmacological activity or other direct effect in the diagnosis, cure,mitigation, treatment, or prevention of disease.

The term “pharmaceutical composition” shall mean a compositioncomprising at least one active ingredient, whereby the composition isamenable to investigation for a specified, efficacious outcome in amammal.

The term “dosage form” shall mean the physical form in which a drug isproduced and dispensed, such as a tablet, capsule, or an injectable.

The term “obesity,” as used herein, is defined as a body-mass index(BMI) of 30.0 or greater, in accordance with the WHO classifications ofweight [Kopelman, Nature (2000) 404:635-643; the disclosure of which isherein incorporated by reference in its entirety].

The term “condition related to obesity” is intended to include but notbe limited to hypertension, congestive cardiomyopathy, varicosities,pulmonary embolism, coronary heart disease, stroke, idiopathicintracranial hypertension, meralgia parethetica, dyspnea, obstructivesleep apnea, hypoventilation syndrome, Pickwickian syndrome, asthma,immobility, degenerative osteoarthritis, low back pain, striae distensaeor “stretch marks,” venous stasis of the lower extremities, lymphedema,cellulitis, intertrigo, carbuncles, acanthosis nigricans, skin tags,gastro-esophageal reflux disorder, nonalcoholic fattyliver/steatohepatitis, cholelithiasis, hernias, colon cancer, stressincontinence, obesity-related glomerulopathy, breast and uterine cancer,depression and low self-esteem, impaired quality of life, metabolicsyndrome, Type 2 diabetes, dyslipidemia, hyperandrogenemia in women,polycystic ovarian syndrome, dysmenorrhea, infertility, pregnancycomplications, and male hypogonadism, where it is understood thatconditions related to obesity can be included in embodimentsindividually or in any combination.

The term “overweight,” as used herein, is defined as a body mass index(BM) of 27-29.9.

As used herein, the term “diabetes” encompasses both insulin-dependentdiabetes mellitus (also known as Type 1 diabetes) andnon-insulin-dependent diabetes mellitus (also known as Type 2 diabetes).

The term “condition related to diabetes” is intended to include but notbe limited to hyperglycemia, impaired glucose tolerance, insulinresistance, pancreatic beta-cell insufficiency, enteroendocrine cellinsufficiency, glucosuria, metabolic acidosis, cataracts, diabeticnephropathy, diabetic neuropathy, diabetic retinopathy, diabeticcoronary artery disease, diabetic cerebrovascular disease, diabeticperipheral vascular disease, metabolic syndrome, hyperlipidemia,atherosclerosis, stroke, hypertension, and obesity, where it isunderstood that conditions related to diabetes can be included inembodiments individually or in any combination.

The term “condition ameliorated by increasing a blood GLP-1 level” isintended to include but not be limited to diabetes, a condition relatedto diabetes, myocardial infarction, learning impairment, memoryimpairment, and a neurodegenerative disorder, where it is understoodthat conditions ameliorated by increasing a blood GLP-1 level can beincluded in embodiments individually or in any combination.

The term “atherosclerosis” as used herein refers to a form of vasculardisease characterized by the deposition of atheromatous plaquescontaining cholesterol and lipids on the innermost layer of the walls oflarge and medium-sized arteries.

The term “metabolic syndrome” as defined herein, and according to theAdult Treatment Panel III (ATP m; National Institutes of Health: ThirdReport of the National Cholesterol Education Program Expert Panel onDetection, Evaluation, and Treatment of High Blood Cholesterol in Adults(Adult Treatment Panel III), Executive Summary; Bethesda, Md., NationalInstitutes of Health, National Heart, Lung and Blood Institute, 2001(NIH pub. No 01-3670), occurs when a person meets three or more of fivecriteria related to obesity, hypertriglyceridemia, low HDL cholesterol,high blood pressure, and high fasting glucose.

The term “neurodegenerative disorder” is intended to include but not belimited to excitotoxic brain damage caused by severe epileptic seizures,Alzheimer's disease, Parkinson's disease, Huntington's disease,prion-associated disease, stroke, motor-neuron disease, learning ormemory impairment, traumatic brain injury, spinal cord injury, andperipheral neuropathy.

Chemical Group, Moiety or Radical

The term “C₁₋₅ acyl” denotes a C₁₋₅ alkyl radical attached to a carbonylwherein the definition of alkyl has the same definition as describedherein; some examples include but not limited to, acetyl, propionyl,n-butanoyl, iso-butanoyl, sec-butanoyl, t-butanoyl (i.e., pivaloyl),pentanoyl and the like.

The term “C₁₋₅ acyloxy” denotes an acyl radical attached to an oxygenatom wherein acyl has the same definition has described herein; someexamples include but not limited to acetyloxy, propionyloxy,butanoyloxy, iso-butanoyloxy, sec-butanoyloxy, t-butanoyloxy and thelike.

The term “C₁₋₆ acylsulfonamide” refers to a C₁₋₆ acyl attached directlyto the nitrogen of the sulfonamide, wherein the definitions for C₁₋₆acyl and sulfonamide have the same meaning as described herein, and aC₁₋₆ acylsulfonamide can be represented by the following formula:

Some embodiments of the present invention are when acylsulfonamide is aC₁₋₅ acylsulfonamide, some embodiments are C₁₋₄ acylsulfonamide, someembodiments are C₁₋₃ acylsulfonamide, and some embodiments are C₁₋₂acylsulfonamide. Examples of an acylsulfonamide include, but not limitedto, acetylsulfamoyl [—S(═O)₂NHC(═O)Me], propionylsulfamoyl[—S(═O)₂NHC(═O)Et], isobutyrylsulfamoyl, butyrylsulfamoyl,2-methyl-butyrylsulfamoyl, 3-methyl-butrylsulfamoyl,2,2-dimethyl-propionylsulfamoyl, pentanoylsulfamoyl,2-methyl-pentanoylsulfamoyl, 3-methyl-pentanoylsulfamoyl,4-methyl-pentanoylsulfamoyl, and the like.

The term “C₂₋₆ alkenyl” denotes a radical containing 2 to 6 carbonswherein at least one carbon-carbon double bond is present, someembodiments are 2 to 4 carbons, some embodiments are 2 to 3 carbons, andsome embodiments have 2 carbons. Both E and Z isomers are embraced bythe term “alkenyl.” Furthermore, the term “alkenyl” includes di- andtri-alkenyls. Accordingly, if more than one double bond is present thenthe bonds may be all E or Z or a mixtures of E and Z. Examples of analkenyl include vinyl, allyl, 2-butenyl, 3-butenyl, 2-pentenyl,3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexanyl,2,4-hexadienyl and the like.

The term “C₁₋₄ alkoxy” as used herein denotes a radical alkyl, asdefined herein, attached directly to an oxygen atom. Examples includemethoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, t-butoxy, iso-butoxy,sec-butoxy and the like.

The term “C₁₋₈ alkyl” denotes a straight or branched carbon radicalcontaining 1 to 8 carbons, some embodiments are 1 to 6 carbons, someembodiments are 1 to 3 carbons, and some embodiments are 1 or 2 carbons.Examples of an alkyl include, but not limited to, methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, t-butyl, pentyl,iso-pentyl, t-pentyl, neo-pentyl, 1-methylbutyl [i.e.,—CH(CH₃)CH₂CH₂CH₃], 2-methylbutyl [i.e., —CH₂CH(CH₃)CH₂CH₃], n-hexyl andthe like.

The term “C₁₋₄ alkylcarboxamido” or “C₁₋₄ alkylcarboxamide” denotes asingle C₁₋₄ alkyl group attached to the nitrogen of an amide group,wherein alkyl has the same definition as found herein. The C₁₋₅alkylcarboxamido may be represented by the following:

Examples include, but not limited to, N-methylcarboxamide,N-ethylcarboxamide, N-n-propylcarboxamide, N-iso-propylcarboxamide,N-n-butylcarboxamide, N-sec-butylcarboxamide, N-iso-butylcarboxamide,N-t-butylcarboxamide and the like.

The term “C₁₋₃ alkylene” refers to a C₁₋₃ divalent straight carbongroup. In some embodiments C₁₋₃ alkylene refers to, for example, —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, and the like. In some embodiments, C₁₋₃ alkylenerefers to —CH—, —CHCH₂—, —CHCH₂CH₂—, and the like wherein these examplesrelate generally to “A”.

The term “C₁₋₄ alkylsulfinyl” denotes a C₁₋₄ alkyl radical attached to asulfoxide radical of the formula: —S(O)— wherein the alkyl radical hasthe same definition as described herein. Examples include, but notlimited to, methylsulfinyl, ethylsulfinyl, n-propylsulfinyl,iso-propylsulfinyl, n-butylsulfinyl, sec-butylsulfinyl,iso-butylsulfinyl, t-butyl, and the like.

The term “C₁₋₄ alkylsulfonamide” refers to the groups

wherein C₁₋₄ alkyl has the same definition as described herein.

The term “C₁₋₄ alkylsulfonyl” denotes a C₁₋₄ alkyl radical attached to asulfone radical of the formula: —S(O)₂— wherein the alkyl radical hasthe same definition as described herein. Examples include, but notlimited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl,iso-propylsulfonyl, n-butylsulfonyl, sec-butylsulfonyl,iso-butylsulfonyl, t-butyl, and the like.

The term “C₁₋₄ alkylthio” denotes a C₁₋₄ alkyl radical attached to asulfide of the formula: —S— wherein the alkyl radical has the samedefinition as described herein. Examples include, but not limited to,methylsulfanyl (i.e., CH₃S—), ethylsulfanyl, n-propylsulfanyl,iso-propylsulfanyl, n-butylsulfanyl, sec-butylsulfanyl,iso-butylsulfanyl, t-butyl, and the like.

The term “C₁₋₄ alkylthiocarboxamide” denotes a thioamide of thefollowing formulae:

wherein C₁₋₄ alkyl has the same definition as described herein.

The term “C₁₋₄ alkylthioureyl” denotes the group of the formula:

—NC(S)N— wherein one are both of the nitrogens are substituted with thesame or different C₁₋₄ alkyl groups and alkyl has the same definition asdescribed herein. Examples of an alkylthioureyl include, but not limitedto, CH₃NHC(S)NH—, NH₂C(S)NCH₃—, (CH₃)₂N(S)NH—, (CH₃)₂N(S)NH—,(CH₃)₂N(S)NCH₃—, CH₃CH₂NHC(S)NH—, CH₃CH₂NHC(S)NCH₃—, and the like.

The term “C₁₋₄ alkylureyl” denotes the group of the formula: —NC(O)N—wherein one are both of the nitrogens are substituted with the same ordifferent C₁₋₄ alkyl group wherein alkyl has the same definition asdescribed herein. Examples of an alkylureyl include, but not limited to,CH₃NHC(O)NH—, NH₂C(O)NCH₃—, (CH₃)₂N(O)NH—, (CH₃)₂N(O)NH—,(CH₃)₂N(O)NCH₃—, CH₃CH₂NHC(O)NH—, CH₃CH₂NHC(O)NCH₃—, and the like.

The term “C₂₋₆ alkynyl” denotes a radical containing 2 to 6 carbons andat least one carbon-carbon triple bond, some embodiments are 2 to 4carbons, some embodiments are 2 to 3 carbons, and some embodiments have2 carbons. Examples of an alkynyl include, but not limited to, ethynyl,1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl,2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl,4-hexynyl, 5-hexynyl and the like. The term “alkynyl” includes di- andtri-ynes.

The term “amino” denotes the group —NH₂.

The term “C₁₋₄ alkylamino” denotes one alkyl radical attached to anamino radical wherein the alkyl radical has the same meaning asdescribed herein. Some examples include, but not limited to,methylamino, ethylamino, n-propylamino, iso-propylamino, n-butylamino,sec-butylamino, iso-butylamino, t-butylamino, and the like. Someembodiments are “C₁₋₂ alkylamino.”

The term “aryl” denotes an aromatic ring radical containing 6 to 10 ringcarbons. Examples include phenyl and naphthyl.

The term “arylalkyl” defines a C₁-C₄ alkylene, such as —CH₂—, —CH₂CH₂—and the like, which is further substituted with an aryl group. Examplesof an “arylalkyl” include benzyl, phenethylene and the like.

The term “arylcarboxamido” denotes a single aryl group attached to thenitrogen of an amide group, wherein aryl has the same definition asfound herein. The example is N-phenylcarboxamide.

The term “arylureyl” denotes the group —NC(O)N— where one of thenitrogens are substituted with an aryl.

The term “benzyl” denotes the group —CH₂C₆H₅.

The term “carbo-C₁₋₆alkoxy” refers to a C₁₋₆ alkyl ester of a carboxylicacid, wherein the alkyl group is as defined herein. In some embodiments,the carbo-C₁₋₆-alkoxy group is bonded to a nitrogen atom and togetherform a carbamate group (e.g., N—COO—C₁₋₆-alkyl). In some embodiments,the carbo-C₁₋₆-alkoxy group is an ester (e.g., —COO—C₁₋₆-alkyl).Examples include, but not limited to, carbomethoxy, carboethoxy,carbopropoxy, carboisopropoxy, carbobutoxy, carbo-sec-butoxy,carbo-iso-butoxy, carbo-t-butoxy, carbo-n-pentoxy, carbo-iso-pentoxy,carbo-t-pentoxy, carbo-neo-pentoxy, carbo-n-hexyloxy, and the like.

The term “carboxamide” refers to the group —CONH₂.

The term “carboxy” or “carboxyl” denotes the group —CO₂H; also referredto as a carboxylic acid group.

The term “cyano” denotes the group —CN.

The term “C₃₋₇ cycloalkenyl” denotes a non-aromatic ring radicalcontaining 3 to 6 ring carbons and at least one double bond; someembodiments contain 3 to 5 carbons; some embodiments contain 3 to 4carbons. Examples include cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentenyl, cyclohexenyl, and the like.

The term “C₃₋₇ cycloalkyl” denotes a saturated ring radical containing 3to 6 carbons; some embodiments contain 3 to 5 carbons; some embodimentscontain 3 to 4 carbons. Examples include cyclopropyl, cyclobutyl,cyclopentyl, cyclopenyl, cyclohexyl, cycloheptyl and the like.

The term “C₄₋₈ diacylamino” denotes an amino group bonded with two acylgroups defined herein wherein the acyl groups may be the same ordifferent, such as:

Examples of C₄₄ diacylamino groups include, but limited to,diacetylamino, dipropionylamino, acetylpropionylamino and the like.

The term “C₂₋₆ dialkylamino” denotes an amino substituted with two ofthe same or different alkyl radicals wherein alkyl radical has the samedefinition as described herein. Some examples include, but not limitedto, dimethylamino, methylethylamino, diethylamino, methylpropylamino,methylisopropylamino, ethylpropylamino, ethylisopropylamino,dipropylamino, propylisopropylamino and the like. Some embodiments are“C₂₋₄ dialkylamino.”

The term “C₁₋₄ dialkylcarboxamido” or “C₁₋₄ dialkylcarboxamide” denotestwo alkyl radicals, that are the same or different, attached to an amidegroup, wherein alkyl has the same definition as described herein. A C₁₋₄dialkylcarboxamido may be represented by the following groups:

wherein C₁₋₄ has the same definition as described herein. Examples of adialkylcarboxamide include, but not limited to, N,N-dimethylcarboxamide,N-methyl-N-ethylcarboxamide, N,N-diethylcarboxamide,N-methyl-N-isopropylcarboxamide, and the like.

The term “C₂₋₆ dialkylsulfonamide” refers to one of the following groupsshown below:

wherein C₁₋₃ has the same definition as described herein, for examplebut not limited to, methyl, ethyl, n-propyl, isopropyl, and the like.

The term “C₂₋₆ dialkylthiocarboxamido” or “C₂₋₆ dialkylthiocarboxamide”denotes two alkyl radicals, that are the same or different, attached toa thioamide group, wherein alkyl has the same definition as describedherein. A C₁₋₄ dialkylthiocarboxamido may be represented by thefollowing groups:

Examples of a dialkylthiocarboxamide include, but not limited to,N,N-dimethylthiocarboxamide, N-methyl-N-ethylthiocarboxamide and thelike.

The term “C₂₋₆ dialkylsulfonylamino” refers to an amino group bondedwith two C₁₋₃ alkylsulfonyl groups as defined herein.

The term “ethynylene” refers to the carbon-carbon triple bond group asrepresented below:

The term “formyl” refers to the group —CHO.

The term “C₁₋₄ haloalkoxy” denotes a haloalkyl, as defined herein, whichis directly attached to an oxygen atonl Examples include, but notlimited to, difluoromethoxy, trifluoromethoxy, 2,2,2-trifluoroethoxy,pentafluoroethoxy and the like.

The term “C₁₋₄ haloalkyl” denotes an C₁₋₄ alkyl group, defined herein,wherein the alkyl is substituted with one halogen up to fullysubstituted and a fully substituted C₁₋₄ haloalkyl can be represented bythe formula C_(n)L_(2n+1) wherein L is a halogen and “n” is 1, 2, 3 or4; when more than one halogen is present then they may be the same ordifferent and selected from the group consisting of F, Cl, Br and I,preferably F. Examples of C₁₋₄ haloalkyl groups include, but not limitedto, fluoromethyl, difluoromethyl, trifluoromethyl, chlorodifluoromethyl,2,2,2-trifluoroethyl, pentafluoroethyl and the like.

The term “C₁₋₄ haloalkylcarboxamide” denotes an alkylcarboxamide group,defined herein, wherein the alkyl is substituted with one halogen up tofully substituted represented by the formula C_(n)L_(2n+1) wherein L isa halogen and “n” is 1, 2, 3 or 4. When more than one halogen is presentthey may be the same or different and selected from the group consistingof F, Cl, Br and I, preferably F.

The term “C₁₋₄ haloalkylsulfinyl” denotes a haloalkyl radical attachedto a sulfoxide group of the formula: —S(O)— wherein the haloalkylradical has the same definition as described herein. Examples include,but not limited to, trifluoromethylsulfinyl,2,2,2-trifluoroethylsulfinyl, 2,2-difluoroethylsulfinyl and the like.

The term “C₁₋₄ haloalkylsulfonyl” denotes a haloalkyl radical attachedto a sulfone group of the formula: —S(O)₂— wherein haloalkyl has thesame definition as described herein. Examples include, but not limitedto, trifluoromethylsulfonyl, 2,2,2-trifluoroethylsulfonyl,2,2-difluoroethylsulfonyl and the like.

The term “C₁₋₄ haloalkylthio” denotes a haloalkyl radicaol directlyattached to a sulfur wherein the haloalkyl has the same meaning asdescribed herein. Examples include, but not limited to,trifluoromethylthio (i.e., CF₃S—), 1,1-difluoroethylthio,2,2,2-trifluoroethylthio and the like.

The term “halogen” or “halo” denotes to a fluoro, chloro, bromo or iodogroup.

The term “C₁₋₂ heteroalkylene” refers to a C₁₋₂ alkylene bonded to aheteroatom selected from O, S, S(O), S(O)₂ and NH. Some representedexamples include, but not limited to, the groups of the followingformulae:

and the like.

The term “heteroaryl” denotes an aromatic ring system that may be asingle ring, two fused rings or three fused rings wherein at least onering carbon is replaced with a heteroatom selected from, but not limitedto, the group consisting of O, S and N wherein the N can be optionallysubstituted with H, C₁₋₄ acyl or C₁₋₄ alkyl. Examples of heteroarylgroups include, but not limited to, pyridyl, benzofuranyl, pyrazinyl,pyridazinyl, pyrimidinyl, triazinyl, quinoline, benzoxazole,benzothiazole, 1H-benzimidazole, isoquinoline, quinazoline, quinoxalineand the like. In some embodiments, the heteroaryl atom is O, S, NH,examples include, but not limited to, pyrrole, indole, and the like.

The term “heterocyclic” denotes a non-aromatic carbon ring (i.e.,cycloalkyl or cycloalkenyl as defined herein) wherein one, two or threering carbons are replaced by a heteroatom selected from, but not limitedto, the group consisting of O, S, N, wherein the N can be optionallysubstituted with H, C₁₋₄ acyl or C₁₋₄ alkyl, and ring carbon atomsoptionally substituted with oxo or a thiooxo thus forming a carbonyl orthiocarbonyl group. The heterocyclic group is a 3-, 4-, 5-, 6- or7-membered containing ring. Examples of a heterocyclic group include butnot limited to aziridin-1-yl, aziridin-2-yl, azetidin-1-yl,azetidin-2-yl, azetidin-3-yl, piperidin-1-yl, piperidin-4-yl,morpholin-4-yl, piperzin-4-yl, piperzin-4-yl, pyrrolidin-1-yl,pyrrolidin-3-yl, [1,3]-dioxolan-2-yl and the like.

The term “heterocyclic-carbonyl” denotes a heterocyclic group, asdefined herein, directly bonded to the carbon of a carbonyl group (i.e.,C═O). In some embodiments, a ring nitrogen of the heterocyclic group isbonded to the carbonyl group forming an amide. Examples include, but notlimited to,

and the like.

In some embodiments, a ring carbon is bonded to the carbonyl groupforming a ketone group. Examples include, but not limited to,

and the like.

The term “heterocyclic-oxy” refers to a heterocyclic group, as definedherein, that is directly bonded to an oxygen atom. Examples include thefollowing:

and the like.

The term “heterocycliccarboxamido” denotes a heterocyclic group, asdefined herein, with a ring nitrogen where the ring nitrogen is bondeddirectly to the carbonyl forming an amide. Examples include, but notlimited to,

and the like.

The term “heterocyclicsulfonyl” denotes a heterocyclic group, as definedherein, with a ring nitrogen where the ring nitrogen is bonded directlyto an SO₂ group forming an sulfonamide. Examples include, but notlimited to,

and the like.

The term “hydroxyl” refers to the group —OH.

The term “hydroxylamino” refers to the group —NHOH.

The term “nitro” refers to the group —NO₂.

The term “C₄₋₇ oxo-cycloalkyl” refers to a C₄₋₇ cycloalkyl, as definedherein, wherein one of the ring carbons is replaced with a carbonyl.Examples of C₄₋₇ oxo-cycloalkyl include, but are not limited to,2-oxo-cyclobutyl, 3-oxo-cyclobutyl, 3-oxo-cyclopentyl, 4-oxo-cyclohexyl,and the like and represented by the following structures respectively:

The term “perfluoroalkyl” denotes the group of the formula—C_(n)F_(2n+1); stated differently, a perfluoroalkyl is an alkyl asdefined herein wherein the alkyl is fully substituted with fluorineatoms and is therefore considered a subset of haloalkyl. Examples ofperfluoroalkyls include CF₃, CF₂CF₃, CF₂CF₂CF₃, CF(CF₃)₂, CF₂CF₂CF₂CF₃,CF₂CF(CF₃)₂, CF(CF₃)CF₂CF₃ and the like.

The term “phenoxy” refers to the group C₆H₅O—.

The term “phenyl” refers to the group C₆H₅—.

The term “phosphonooxy” refers to a group with the following chemicalstructure:

The term “sulfonamide” refers to the group —SO₂NH₂.

The term “sulfonic acid” refers to the group —SO₃H.

The term “tetrazolyl” refers to the five membered heteroaryl of thefollowing formulae:

In some embodiments, the tetrazolyl group is further substituted ateither the 1 or 5 position respectively with a group selected from thegroup consisting of C₁₋₃ alkyl, C₁₋₃ haloalkyl and C₁₋₃ alkoxy.

The term “thiol” denotes the group —SH.

The term “Apa” refers to amino-3-phenylpropionic acid.

The term “GLP-1 secretagogue” shall mean an agent (e.g., a compound)that promotes GLP-1 secretion from a cell, e.g. an enteroendocrine cell.

The term “endogenous” shall mean a material that a mammal naturallyproduces.

The term “biologically active fragment of a G protein-coupled receptor”shall mean a fragment of the GPCR having structural and biochemicalfunctions of a naturally occurring GPCR. In certain embodiments, thebiologically active fragment couples to a G protein. In certainembodiments, the biologically active fragment binds to a known ligand ofthe GPCR.

The term “primer” is used herein to denote a specific oligonucleotidesequence which is complementary to a target nucleotide sequence and usedto hybridize to the target nucleotide sequence. A primer serves as aninitiation point for nucleotide polymerization catalyzed by DNApolymerase, RNA polymerase, or reverse transcriptase.

The term “expression vector” shall mean a DNA sequence that is requiredfor the transcription of cloned DNA and translation of the transcribedmRNA in an appropriate host cell recombinant for the expression vector.An appropriately constructed expression vector should contain an originof replication for autonomous replication in host cells, selectablemarkers, a limited number of useful restriction enzyme sites, apotential for high copy number, and active promoters. The cloned DNA tobe transcribed is operably linked to a constitutively or conditionallyactive promoter within the expression vector.

The term “candidate compound” or “test compound” shall mean a compound(for example and not limitation, a chemical compound) that is amenableto screening.

The term “contact” or “contacting” shall mean bringing at least twomoieties together.

The terms “modulate” or “modify” shall be taken to refer to an increaseor decrease in the amount, quality, or effect of a particular activity,function or molecule. By way of illustration and not limitation,agonists, partial agonists, inverse agonists, and antagonists of a Gprotein-coupled receptor are modulators of the receptor.

The term “small molecule” shall be taken to mean a compound having amolecular weight of less than about 10,000 grams per mole, including apeptide, peptidomimetic, amino acid, amino acid analogue,polynucleotide, polynucleotide analogue, nucleotide, nucleotideanalogue, organic compound or inorganic compound (I.e. including aheterorganic compound or organometallic compound), and salts, esters andother pharmaceutically acceptable forms thereof. In certain preferredembodiments, small molecules are organic or inorganic compounds having amolecular weight of less than about 5,000 grams per mole. In certainpreferred embodiments, small molecules are organic or inorganiccompounds having molecular weight of less than about 1,000 grams permole. In certain preferred embodiments, small molecules are organic orinorganic compounds having a molecular weight of less than about 500grams per mole.

The term “polynucleotide” shall refer to RNA, DNA, or RNA/DNA hybridsequence of more than one nucleotide in either single chain or duplexform. The polynucleotides of the invention may be prepared by any knownmethod, including synthetic, recombinant, ex vivo generation, or acombination thereof, as well as utilizing any purification methods knownin the art.

The term “polypeptide” shall refer to a polymer of amino acids withoutregard to the length of the polymer. Thus, peptides, oligopeptides, andproteins are included within the definition of polypeptide. This termalso does not specify or exclude post-expression modifications ofpolypeptides. For example, polypeptides that include the covalentattachment of glycosyl groups, acetyl groups, phosphate groups, lipidgroups and the like are expressly encompassed by the term polypeptide.

The term “antibody” is intended herein to encompass monoclonal antibodyand polyclonal antibody.

The term “second messenger” shall mean an intracellular responseproduced as a result of receptor activation. A second messenger caninclude, for example, inositol 1,4,5-triphosphate (IP3), diacylglycerol(DAG), cyclic AMP (cAMP), cyclic GMP (cGMP), MAP kinase activity,MAPK/ERK kinase kinase-1 (MEKK1) activity, and Ca2+. Second messengerresponse can be measured for a determination of receptor activation.

The term “receptor functionality” shall refer to the normal operation ofa receptor to receive a stimulus and moderate an effect in the cell,including, but not limited to regulating gene transcription, regulatingthe influx or efflux of ions, effecting a catalytic reaction, and/ormodulating activity through G-proteins, such as eliciting a secondmessenger response.

The term “stimulate” or “stimulating,” in relationship to the term“response” or “functionality of the receptor” shall mean that a responseor a functionality of the receptor is increased in the presence of acompound as opposed to in the absence of the compound.

The term “inhibit” or “inhibiting,” in relationship to the term“response” or “functionality of the receptor” shall mean that a responsea functionality of the receptor is decreased or prevented in thepresence of a compound as opposed to in the absence of the compound.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the lower limit unless the contextclearly indicates otherwise, between the upper and lower limit of thatrange and any other stated or intervening value in that stated range, isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

BRS-3 Agonists

Preferably, BRS-3 is mammalian BRS-3. More preferably, BRS-3 is rodentor primate BRS-3. Most preferably, BRS-3 is human BRS-3.

The class of BRS-3 agonists useful in the novel therapeutic combinationsof the present invention include compounds which exhibit an acceptablyhigh affinity for BRS-3 receptor. The BRS-3 agonist or pharmaceuticallyacceptable salt may be any agonist, more preferably a selective BRS-3agonist.

Examples of BRS-3 agonists are described in Weber et al., J Med Chem(2003) 46:1918-1930, the disclosure of which is herein incorporated byreference in its entirety.

Examples of BRS-3 agonists are described in Mantey et al., J PharmacolExp Ther (2004) 310:1161-1169, the disclosure of which is hereinincorporated by reference in its entirety.

Examples of BRS-3 agonists are described in Boyle et al., J Peptide Sci(2005) 11:136-141, the disclosure of which is herein incorporated byreference in its entirety.

Examples of BRS-3 agonists are described in Lamerich et al., Br JPharmacol (2003) 138:1431-1440, the disclosure of which is hereinincorporated by reference in its entirety.

In one aspect, the BRS-3 agonist is selected from the left column ofTable B.

In one aspect, the BRS-3 agonist is selected from Compound 1, Compound2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13,Compound 14, Compound 15, Compound 16, Compound 17, Compound 18,Compound 19, Compound 20, Compound 21, Compound 22, Compound 23,Compound 24, Compound 25, Compound 26, and Compound 27; these Compoundsmay be found in Boyle et al., J Peptide Sci (2005) 11:136-141, wherethey are described as selective BRS-3 agonists.

In one aspect, the BRS-3 agonist is selected from Compound 28, Compound29, and Compound 30; these Compounds may be found in Mantey et al., JPharmacol Exp Ther (2004) 310:1161-1169, where they are described asselective BRS-3 agonists.

In one aspect, the BRS-3 agonist is selected from Compound 31, Compound32, and Compound 33; these Compounds may be found in Boyle et al., JPeptide Sci (2005) 11:136-141, where they are described as selectiveBRS-3 agonists.

In one aspect, the BRS-3 agonist is Compound 34; this Compound may befound in Mantey et al., J Pharmacol Exp Ther (2004) 310:1161-1169, whereit is described as non-selective for BRS-3 over GRP-R and NMB-R

In one aspect of the present invention, any one or more BRS-3 agonistcan be excluded from any embodiment of the present invention.

In one aspect of the present invention, the BRS-3 agonist has an EC50 ofless than about 10 μM, less than about 1 μM, less than about 100 nM,less than about 75 nM, less than about 50 nM, less than about 25 nM,less than about 20 nM, less than about 15 nM, less than about 10 nM,less than about 5 nM, less than about 4 nM, less than about 3 nM, lessthan about 2 nM, or less than about 1 nM. Preferably the BRS-3 agonisthas an EC50 of less than about 50 nM, less than about 25 nM, less thanabout 20 nM, less than about 15 nM, less than about 10 nM, less thanabout 5 nM, less than about 4 nM, less than about 3 nM, less than about2 nM, or less than about 1 nM.

In one aspect of the present invention, the BRS-3 agonist is a selectiveBRS-3 agonist, wherein the selective BRS-3 agonist has a selectivity forBRS-3 over gastric-releasing peptide receptor (GRP-R) or neuromedin Breceptor (NMB-R) of at least about 10-fold, more preferably of at leastabout 100-fold. In one aspect of the present invention, the BRS-3agonist is a selective BRS-3 agonist, wherein the selective BRS-3agonist has a selectivity for BRS-3 over gastric-releasing peptidereceptor (GRP-R) and neuromedin B receptor (NMB-R) of at least about10-fold, more preferably of at least about 100-fold.

In one aspect of the present invention, the BRS-3 agonist is orallyactive.

In one aspect of the present invention, the BRS-3 agonist is an agonistof human BRS-3.

DPP-IV Inhibitors

The class of DPP-IV inhibitors useful in the novel therapeuticcombinations of the present invention include compounds which exhibit anacceptably high affinity for DPP-IV. The DPP-IV inhibitor orpharmaceutically acceptable salt may be any DPP-IV inhibitor, morepreferably a selective dipeptidyl peptidase inhibitor, and mostpreferably a selective DPP-IV inhibitor.

Examples of DPP-IV inhibitors are described in Villhauer et al., J MedChem (2003) 46:2774-2789, for LAF237; Ahren et al, J Clin EndocrinolMetab (2004) 89:2078-2084; Villhauer et al., J Med Chem (2002)45:2362-2365 for NVP-DPP728; Abren et al, Diabetes Care (2002)25:869-875 for NVP-DPP728; Peters et al., Bioorg Med Chem Lett (2004)14:1491-1493; Caldwell et al., Bioorg Med Chem Lett (2004) 14:1265-1268;Edmondson et al., Bioorg Med Chem Lett (2004) 14:5151-5155; and Abe etal., J Nat Prod (2004) 67:999-1004; the disclosure of each of which isherein incorporated by reference in its entirety.

Specific examples of DPP-IV inhibitors include, but are not limited to,dipeptide derivatives or dipeptide mimetics such as alanine-pyrrolidide,isoleucine-thiazolidide, and the pseudosubstrate N-valyl prolyl,O-benzoyl hydroxylamine, as described e.g. in U.S. Pat. No. 6,303,661,the disclosure of which is herein incorporated by reference in itsentirety.

Examples of DPP-IV inhibitors may be found in U.S. Pat. Nos. 6,812,350,6,803,357, 6,710,040, 6,617,340, 6,699,871, 6,573,287, 6,432,969,6,395,767, 6,303,661, 6,242,422, 6,166,063, the disclosure of each ofwhich is herein incorporated by reference in its entirety. Examples ofDPP-IV inhibitors may be found in U.S. Pat. Appl. Nos. 2004242898,2004180925, 2004110817, 2004106656, 2003232788, 2003216450, 2003134802,2003125304, 2003130281, 2003119738, 2003100563, 2003119750, 2003130199,2002183367, 2002049164, 2002006899, the disclosure of each of which isherein incorporated by reference in its entirety.

Examples of DPP-IV inhibitors may be found in International ApplicationsWO 04/103993, WO 04/103276, WO 04/99134, WO 04/87053, WO 04/76434, WO04/76433, WO 04/69162, WO 04/64778, WO 04/71454, WO 04/69162, WO04/67509, WO 04/58266, WO 04/52850, WO 04/50022, WO 04/50658, WO04/32836, WO 04/46106, WO 04/43940, WO 04/41795, WO 04/37169, WO04/37181, WO 03/101958, WO 04/14860, WO 04/07468, WO 04/04661, WO03/82817, WO 03/72528, WO 03/57666, WO 03/57144, WO 03/40174, WO03/37327, WO 03/35067, WO 03/35057, WO 03/22871, WO 03/15775, WO03/04498, WO 03/02530, WO 03/02596, WO 03/02595, WO 03/02593, WO03/02553, WO 03/02531, WO 03/00181, WO 03/00180, WO 03/00250, WO02/83109, WO 02/83128, WO 02/76450, WO 02/51836, WO 02/34900, WO01/96295, WO 01/81337, WO 01/81304, WO 01/68603 WO 01/34594, WO00/34241, WO 00/23421, WO 99/67278, WO 99/61431, WO 98/19998, WO97/40832, EP 1480961, EP 1469873, EP 1465891, EP 1450794, EP 1446116, EP1412357, EP 1399420, EP 1399471, EP 1399470, EP 1399469, EP 1399433,EP1406873, EP 1406622, EP 1406872, EP 1399154, EP 1377288, EP 1385508,EP 1354882, EP 1304327, EP 1296974, EP 1280797, EP 1282600, EP 1261586,EP 1215207, EP 1228061, EP 1137635, EP 1123272, EP 1082314, CA 2433090,DE 19828113, DE 19823831, DE 19616486, DE 10256264, DE 10143840, JP2004244412, JP 2004026820, JP 2003300977, JP 2004521149, JP 2004530729,JP 2004525179, JP 2004525929, JP 2004503531, JP 2003535034, JP2003531204, JP 2003531191, JP 2003531118, JP 2003524591, JP 2002531547,JP 2002527504, JP 2002516318, JP 2001510442, JP 2000511559, JP2000191616, the disclosure of each of which is herein incorporated byreference in its entirety.

In one aspect of the present invention, the DPP-IV inhibitor isvaline-pyrrolidide [Deacon et al, Diabetes (1998) 47:764769; thedisclosure of which is herein incorporated by reference in itsentirety].

In one aspect of the present invention, the DPP-IV inhibitor is3-(L-Isoleucyl)thiazolidine (isoleucine-thiazolidide).Isoleucine-thiazolidide may be found in JP 2001510442, WO 97/40832, U.S.Pat. No. 6,303,661, and DE 19616486, the disclosure of each of which isherein incorporated by reference in its entirety.Isoleucine-thiazolidide is described as an orally active and selectiveDPP-IV inhibitor [Pederson et al, Diabetes (1998) 47:1253-1258; thedisclosure of which is herein incorporated by reference in itsentirety].

In one aspect of the present invention, the DPP-IV inhibitor is1-[2-[5-cyanopyridin-2-yl)amino]ethylamino]acetyl-2-cyano-(S)-pyrrolidine(NVP-DPP728). NVP-DPP728 may be found in WO 98/19998 and JP 2000511559,the disclosure of each of which is herein incorporated by reference inits entirety. NVP-DPP728 is described as an orally active and selectiveDPP-IV inhibitor [Villhauer et al, J Med Chem (2002) 45:2362-2365].

In one aspect of the present invention, the DPP-IV inhibitor is3(R)-Amino-1-[3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butan-1-one(MK-0431). MK-0431 maybe found in EP 1412357, WO 03/04498, U.S. Pat. No.6,699,871, and US 2003100563, the disclosure of each of which is hereinincorporated by reference in its entirety. MK-0431 is described as anorally active and selective DPP-IV inhibitor [Weber et al, Diabetes(2004) 53(Suppl. 2):A151, 633-P (Abstract), the disclosure of which isherein incorporated by reference in its entirety].

In one aspect of the present invention, the DPP-IV inhibitor is(1-[[3-hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine(AF237). LAF237 may be found in U.S. Pat. No. 6,166,063, WO 00/34241, EP1137635, and JP 2002531547, the disclosure of each of which is hereinincorporated by reference in its entirety. LAF237 is described as anorally active and selective DPP-IV inhibitor [Villhauer et al, J MedChem (2003) 46:2774-2789].

In one aspect of the present invention, the DPP-IV inhibitor is(1S,3S,5S)-2-[2(S)-Amino-2-(3-hydroxyadamantan-1-yl)acetyl]-2-azabicyclo[3.1.0]hexane-3-carbonitrile(BMS477118).

In one aspect of the present invention, the DPP-IV inhibitor is selectedfrom the right column of Table B.

In one aspect of the present invention, any one or more DPP-IV inhibitorcan be excluded from any embodiment of the present invention.

In one aspect of the present invention, the DPP-IV inhibitor has an IC50of less than about 10 μM, less than about 1 μM, less than about 100 nM,less than about 75 nM, less than about 50 nM, less than about 25 nM,less than about 20 nM, less than about 15 nM, less than about 10 nM,less than about 5 nM, less than about 4 nM, less than about 3 nM, lessthan about 2 nM, or less than about 1 nM. Preferably the DPP-IVinhibitor has an IC50 of less than about 50 nM, less than about 25 nM,less than about 20 nM, less than about 15 nM, less than about 10 nM,less than about 5 nM, less than about 4 nM, less than about 3 nM, lessthan about 2 nM, or less than about 1 nM.

In one aspect of the present invention, the DPP-IV inhibitor a selectiveDPP-IV inhibitor, wherein the selective DPP-IV inhibitor has aselectivity for human plasma DPP-IV over one or more of PPCE, DPP-II,DPP-8 and DPP-9 of at least about 10-fold, more preferably of at leastabout 100-fold, and most preferably of at least about 1000-fold.

In one aspect of the present invention, the DPP-IV inhibitor is orallyactive.

In one aspect of the present invention, the DPP-IV inhibitor is aninhibitor of human DPP-IV.

Combination of BRS-3 Agonist and DPP-IV Inhibitor

By way of illustration and not limitation, an exemplary combination ofBRS-3 agonist and DPP-IV inhibitor in accordance with the presentinvention is provided by selecting a BRS-3 agonist from the left columnof Table B and a DPP-IV inhibitor from the right column of Table B. Itis expressly contemplated that each individual combination of BRS-3agonist and DPP-IV inhibitor provided by selecting a BRS-3 agonist fromthe left column of Table B and a DPP-IV inhibitor from the right columnof Table B is a separate embodiment within the scope of the presentinvention.

TABLE B BRS-3 Agonist DPP-IV InhibitorN1-{(1R)-2-(1H-3-Indolyl)-1-[1-(2- valine-pyrrolididephenylethyl)carbamoyl]ethyl}-(2S)-2-[1-(2-(4-chlorophenyl)ethyl)carboxamido]pentanediamide (Compound 1)N1-{(1R)-2-(1H-3-Indolyl)-1-[1-(2- 3-(L-Isoleucyl)thiazolidinephenylethyl)carbamoyl]ethyl}-(2S)-2-[(1H-2- (isoleucine-thiazolidide)indolylmethyl)carboxamido]pentanediamide (Compound 2)N1-(2-Phenylethyl)-(2R)-2-{[(1S)-1- 1-[2-[5-cyanopyridin-2-(benzylcarboxamido)ethyl]carboxamido}-3-(1H-3-yl)amino]ethylamino]acetyl-2-cyano- indolyl)propanamide (S)-pyrrolidine(Compound 3) (NVP-DPP728) N1-(2-Phenylethyl)-(2R)-2-{[(1S)-1-((4-3(R)-Amino-1-[3- chlorobenzyl)-carboxamido)ethyl]carboxamido}-3-(trifluoromethyl)-5,6,7,8- (1H-3-indolyl)propanamidetetrahydro[1,2,4]triazolo[4,3- (Compound 4) a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butan-1-one (MK-0431)N1-(2-Phenylethyl)-(2R)-2-{[(1S)-1-((1,3- (1-[[3-hydroxy-1-benzodioxol-5- adamantyl)amino]acetyl]-2-cyano-(S)-ylmethyl)carboxamido)ethyl]carboxamido}-3-(1H-3- pyrrolidineindolyl)propanamide (LAF237) (Compound 5)N1-(2-Phenylethyl)-(2R)-2-{[(1S)-1-((3- (1S,3S,5S)-2-[2(S)-Amino-2-pyridyl)methylcarboxamido)ethyl]carboxamido}-3-(3-hydroxyadamantan-1-yl)acetyl]-2- (1H-3-indolyl)propanamideazabicyclo[3.1.0]hexane-3-carbonitrile (Compound 6) (BMS-477118)N1-(2-Phenylethyl)-(2R)-2-{[(1S)-1-((1,2,3,4- [1-[2(S)-Amino-3-tetrahydro-1- methylbutyryl]pyrrolidin-2(R)-isoquinolinyl)methylcarboxamido)ethyl]carboxamido}- yl]boronic acid3-(1H-3-indolyl)propanamide (PT-100) (Compound 7)N1-(2-Phenylethyl)-(2R)-2-{[(1S)-1-((1H2- GSK-823093indolyl)methylcarboxamido)ethyl]carboxamido}-3-(1H-3-indolyl)propanamide (Compound 8) N1-(2-Phenylethyl)-(2R)-2-PSN-9301 {[(benzyl)amino]methylcarboxamido}-3-(1H-3- indolyl)propanamide(Compound 9) N1-(2-Phenylethyl)-(2R)-2-{[(4- T-6666chlorobenzyl)amino]-methylcarboxamido}-3-(1H-3- indolyl)propanamide(Compound 10) N1-(2-Phenylethyl)-(2R)-2-{[(3- SYR-322pyridyl)methylamino]-methylcarboxamido}-3-(1H-3- indolyl)propanamide(Compound 11) N1-(2-Phenylethyl)-(2R)-2-{[1-(2- SYR-619phenylethyl)amino]-methylcarboxamido}-3-(1H-3- indolyl)propanamide(Compound 12) N1-(2-Phenylethyl)-(2R)-2-{[1- CR-14023benzylamino)ethyl]carboxamido}-3-(1H-3- indolyl)propanamide (Compound13) N1-(2-Phenylethyl)-(2R)-2-{[1-((4- CR-14025chlorobenzyl)amino)ethyl]carboxamido}-3-(1H-3- indolyl)propanamide(Compound 14) N1-(2-Phenylethyl)-(2R)-2-{[1-(1-(2- CR-14240phenylethyl)amino)ethyl]carboxamido}-3- (1H-3-indolyl)propanamide(Compound 15) N1-(2-Phenylethyl)-(2R)-2-{{N′- CR-13651(benzoyl)hydrazino]carboxamido}-3-(1H-3- indolyl)propanamide (Compound16) N1-(2-Phenylethyl)-(2R)-2-{{N′-(4- TS-021chlorobenzoyl)-hydrazino]carboxamido}-3-(1H-3- indolyl)propanamide(Compound 17) N1-(2-Phenylethyl)-(2R)-2-{[N′-(2-(3- GRC-8200pyridyl)ethanoyl)hydrazino]carboxamido}-3-(1H-3- indolyl)propanamide(Compound 18) N1-(2-Phenylethyl)-(2R)-2-{{N′-(2-(1,2,3,4- GRC-8116tetrahydro-1- isoquinolinyl)ethanoyl)hydrazino]carboxamido}-3-(1H-3-indolyl)propanamide (Compound 19)N1-(2-Phenylethyl)-(2R)-2-{[N′-(2-(1H-2- FE107542indolyl)ethanoyl)hydrazino]carboxamido}-3-(1H-3- indolyl)propanamide(Compound 20) N1-(2-Phenylethyl)-(2R)-2-{[N′- 4(S)-Fluoro-1-[2-[1-(2-(phenylmethylene)-hydrazino]carboxamido}-3-(1H-3-hydroxyacetyl)-4-methylpiperidin-4- indolyl)propanamideylamino]acetyl]pyrrolidine-2(S)- (Compound 21) carbonitrile fumarateN1-(2-Phenylethyl)-(2R)-2-{[N′-(furan-2- 6-[2-[2-[2(S)-Cyanoazetidin-1-ylmethylene)hydrazino]carboxamido}-3-(1H-3- yl]-2- indolyl)propanamideoxoethylamino]ethylamino]pyridine-3- (Compound 22) carbonitriledihydrochloride N1-(2-Phenylethyl)-(2R)-2-{[N′-N-(5-Chloropyridin-2-yl)-2-[4- (benzyl)hydrazino]carboxamido}-3-(1H-3-[1-[2-(4-cyanothiazolidin-3-yl)-2- indolyl)propanamideoxoethyl]hydrazino]piperidin-1- (Compound 23) yl]acetamidetris(trifluoroacetate) N1-(2-Phenylethyl)-(2R)-2-{[N′-(4-trans-4-[2-[4(R)- chlorobenzyl)hydrazino]carboxamido}-3-(1H-3-Cyanothiazolidin-3-yl]-2- indolyl)propanamide oxoethylamino]-N,N-(Compound 24) dimethylcyclohexanecarboxamide hydrochlorideN1-(2-Phenylethyl)-(2R)-2-{[N′-(2- trans-1-[2-[4-(1,3-Dioxo-2,3-furylmethyl)hydrazino]carboxamido}-3-(1H-3- dihydro-1H-isoindol-2-indolyl)propanamide yl)cyclohexylamino]acetyl]pyrrolidine- (Compound 25)2(S)-carbonitrile hydrochloride N1-(2-Phenylethyl)-(2R)-2-{[4-4,4-Difluoro-1-[2-[exo-8-(2-benzylpiperazino)methyl]carboxamido}-3-(1H-3-pyrimidinyl)-8-azabicyclo[3.2.1]oct-3- indolyl)propanamideylamino]acetyl]pyrrolidine-2(S)- (Compound 26) carbonitrileN1-(2-Phenylethyl)-(2R)-2-{[(4- exo-3-[2-[8-(2-Pyrimidinyl)-8-benzylpiperidino)methyl]carboxamido}-3-(1H-3- azabicyclo[3.2.1]oct-3-indolyl)propanamide ylamino]acetyl]thiazolidine-4(R)- (Compound 27)carbonitrile [D-Tyr⁶,(R)-Apa¹¹,Phe¹³,Nle¹⁴]Bombesin(6-14)3(R)-Amino-1-(8-chloro- (Compound 28) 1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazol-2-yl)-4-(2,5- difluorophenyl)butan-1-one trifluoroacetate[D-Tyr⁶,(S)-Apa¹¹,Phe¹³,Nle¹⁴]Bombesin(6-14) 3(R)-Amino-4-(2,5-(Compound 29) difluorophenyl)-1-[2-(4-fluorophenyl)-4,5,6,7-tetrahydrothiazolo[4,5- c]pyridin-5-yl]butan-1-one[D-Tyr⁶,(R)-Apa¹¹- 2-[4-[2-[3(R)-Amino-4-(2-4Cl,Phe¹³,Nle¹⁴]Bombesin(6-14) fluorophenyl)butyryl]-1,2,3,4- (Compound30) tetrahydroisoquinolin-3- ylcarboxamidomethyl]phenyl]acetic acidAc-Phe-Trp-Ala-His(τBzl)-Nip-Gly-Arg-NH₂ 1-(2-Benzothiazolyl)-1-[1-(Compound 31) [(2S,3aS,7aS)-perhydroindol-2- ylcarbonyl]pyrrolidin-2(S)-yl]methanone hydrochloride Ac-Phe-Trp-Ala-His(τBzl)-βAla-His-Arg-NH₂trans-N-[4-[1(S)-Amino-2- (Compound 32) [3(S)-fluoropyrrolidin-1-yl]-2-oxoethyl]cyclohexyl]-2,4- difluorobenzenesulfonamideAc-Phe-Trp-Ala-Val-βAla-His-Arg-Trp-NH₂ (1S,3S,5S)-2-[2(S)-Amino-4,4-(Compound 33) dimethylpentanoyl]-2- azabicyclo[3.1.0]hexane-3(S)-carbonitrile trifluoroacetate [D-Tyr⁶,βAla¹¹,Phe¹³,Nle¹⁴]Bombesin(6-14)2-[7-(2-Butynyl)-1-(2- (Compound 34)phenylethyl)-8-(1-piperazinyl)xanthin- 3-yl]-N-(2-propynyl)acetamidehydrochloride

Additionally, compounds of the invention, including those illustrated inTABLE B, encompass all pharmaceutically acceptable salts, solvates, andhydrates thereof. See, e.g., Berge et al (1977), Journal ofPharmaceutical Sciences 66:1-19; and Polymorphism in PharmaceuticalSolids (1999) Brittain, ed., Marcel Dekker, Inc.; the disclosure of eachof which is herein incorporated by reference in its entirety.

As relates to the combination therapy described above, the compoundsaccording to the invention can be administered in any suitable way.Suitable routes of administration include oral, nasal, rectal,transmucosal, transdermal, or intestinal administration, parenteraldelivery, including intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, intrapulmonary (inhaled) orintraocular injections using methods known in the art. Other suitableroutes of administration are aerosol and depot formulation. Sustainedrelease formulations, particularly depot, of the invented medicamentsare expressly contemplated. In certain preferred embodiments, thecompounds according to the present invention are administered orally.The compounds according to the present invention can be made up in solidor liquid form, such as tablets, capsules, powders, syrups, elixirs andthe like, aerosols, sterile solutions, suspensions or emulsions, and thelike. In certain embodiments, one or both of the BRS-3 agonist and theDPP-IV inhibitor are administered orally.

Formulations for oral administration may be in the form of aqueoussolutions and suspensions, in addition to solid tablet and capsuleformulations. The aqueous solutions and suspensions may be prepared fromsterile powders or granules. The compounds may be dissolved in water,polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseedoil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/orvarious buffers. Other adjuvants are well and widely known in the art.

It will be appreciated that the BRS-3 agonist and the DPP-IV inhibitormay be present as a combined preparation for simultaneous, separate orsequential use for the treatment or prevention of diabetes or acondition related thereto. Such combined preparations may be, forexample, in the form of a twin pack.

It will therefore be further appreciated that the invention contemplatesa product comprising or consisting essentially of a BRS-3 agonist and aDPP-IV inhibitor as a combined preparation for simultaneous, separate orsequential use in the prevention or treatment of diabetes or a conditionrelated thereto.

A combination of the present invention comprising or consistingessentially of a BRS-3 agonist and a DPP-IV inhibitor can be prepared bymixing the BRS-3 agonist and the DPP-IV inhibitor either all together orindependently with a pharmaceutically acceptable carrier, excipient,binder, dilutent, etc. as described herein, and administering themixture or mixtures either orally or non-orally as a pharmaceuticalcomposition(s).

It will therefore be further appreciated that the BRS-3 agonist and theDPP-IV inhibitor or pharmaceutical composition can be administered inseparate dosage forms or in a single dosage form.

It is further appreciated that when the BRS-3 agonist and the DPP-IVinhibitor are in separate dosage forms, BRS-3 agonist and DPP-IVinhibitor can be administered by different routes.

Pharmaceutical compositions of the BRS-3 agonist and DPP-IV inhibitor,either individually or in combination, may be prepared by methods wellknown in the art, e.g., by means of conventional mixing, dissolving,granulation, dragee-making, levigating, emulsifying, encapsulating,entrapping, lyophilizing processes or spray drying.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Suitablepharmaceutically acceptable carriers are available to those in the art[see, e.g., Remington: The Science and Practice of Pharmacy, (Gennaro etal., eds.), 20th Edition, 2000, Lippincott Williams & Wilkins; andHandbook of Pharmaceutical Excipients (Rowe et al., eds), 4^(th)Edition, 2003, Pharmaceutical Press; the disclosure of each of which isherein incorporated by reference in its entirety]. Proper formulation isdependent upon the route of administration chosen. The term “carrier”material or “excipient” material herein means any substance, not itselfa therapeutic agent, used as a carrier and/or dilutent and/or adjuvant,or vehicle for delivery of a therapeutic agent to a subject or added toa pharmaceutical composition to improve its handling or storageproperties or to permit or facilitate formation of a dose unit of thecomposition into a discrete article such as a capsule or tablet suitablefor oral admininstration. Excipients can include, by way of illustrationand not limitation, diluents, disintegrants, binding agents, adhesives,wetting agents, polymers, lubricants, glidants, substances added to maskor counteract a disagreeable taste or odor, flavors, dyes, fragrances,and substances added to improved appearance of the composition.Acceptable excipients include stearic acid, magnesium stearate,magnesium oxide, sodium and calcium salts of phosphoric and sulfuricacids, magnesium carbonate, talc, gelatin, acacia gum, sodium alginate,pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starches,gelatin, cellulosic materials, such as cellulose esters of alkanoicacids and cellulose alkyl esters, low melting wax cocoa butter orpowder, polymers, such as polyvinyl-pyrrolidone, polyvinyl alcohol, andpolytheylene glycols, and other pharmaceutically acceptable materials.The components of the pharmaceutical composition can be encapsulated ortableted for convenient administration.

Pharmaceutically acceptable refers to those properties and/or substanceswhich are acceptable to the patient from a pharmacological/toxicologicalpoint of view and to the manufacturing pharmaceutical chemist from aphysical/chemical point of view regarding composition, formulation,stability, patient acceptance and bioavailability.

When the BRS-3 agonist and the DPP-IV inhibitor are in separate dosageforms, it is understood that a pharmaceutically acceptable carrier usedfor the BRS-3 agonist formulation need not be identical to apharmaceutically acceptable carrier used for the DPP-IV inhibitorformulation.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum Arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with a fillersuch as lactose, a binder such as starch, and/or a lubricant such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, liquid polyethyleneglycols, cremophor, capmul, medium or long chain mon-, di- ortriglycerides. Stabilizers may be added in these formulations, also.

Additionally, the BRS-3 agonist and DPP-IV inhibitor may be deliveredusing a sustained-release system. Various sustained-release materialshave been established and are well known to those skilled in the art.Sustained-release tablets or capsules are particularly preferred. Forexample, a time delay material such as glyceryl monostearate or glyceryldistearate may be employed. The dosage form may also be coated by thetechniques described in the U.S. Pat. Nos. 4,256,108, 4,166,452, and4,265,874 to form osmotic therapeutic tablets for controlled release.

It is expressly contemplated that a combination therapy of the presentinvention may be administered or provided alone or in combination withone or more other pharmaceutically or physiologically acceptablecompound. In one aspect of the present invention, the otherpharmaceutically or physiologically acceptable compound is not a BRS-3agonist and is not a DPP-IV inhibitor.

In one aspect of the present invention, the other pharmaceutically orphysiologically acceptable compound is a pharmaceutical agent selectedfrom the group consisting of sulfonylurea (e.g., glibenclamide,glipizide, gliclazide, glimepiride), meglitinide (e.g., repaglinide,nateglinide), biguanide (e.g., metformin), alpha-glucosidase inhibitor(e.g., acarbose, epalrestat, miglitol, voglibose), thizaolidinedione(e.g., rosiglitazone, pioglitazone), insulin analog (e.g., insulinlispro, insulin aspart, insulin glargine), chromium picolinatelbiotin,and biological agent (e.g., adiponectin or a fragment comprising theC-terminal globular domain thereof, or a multimer of adiponectin or saidfragment thereof; or an agonist of adiponectin receptor AdipoR1 orAdipoR2, preferably wherein said agonist is orally active). In oneaspect of the present invention, the pharmaceutical agent is metformin.In one aspect of the present invention, the pharmaceutical agent is anagonist to adiponectin receptor AdipoR1 or AdipoR2, preferably whereinthe agonist is orally active.

In one aspect of the present invention, the other pharmaceutically orphysiologically acceptable compound is an anti-obesity agent such asapolipoprotein-B secretion/microsomal triglyceride transfer protein(apo-B/MTP) inhibitors, MCR4 agonists, cholescystokinin-A (CCK-A)agonists, serotonin and norepinephrine reuptake inhibitors (for example,sib nine), sympathomimetic agents, β3 adrenergic receptor agonists,dopamine agonists (for example, bromocriptine), melanocyte-stimulatinghormone receptor analogs, carnabinoid 1 receptor antagonists [forexample, SR141716:N-(piperidin-1-yl)₅₋₄-chlorophenyl)-1-(2,4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide],melanin concentrating hormone antagonists, leptons (the OB protein),leptin analogues, leptin receptor agonists, galanin antagonists, lipaseinhibitors (such as tetrahydrolipstatin, i.e., Orlistat), anorecticagents (such as a bombesin agonist), Neuropeptide-Y antagonists,thyromimetic agents, dehydroepiandrosterone or an analogue thereof,glucocorticoid receptor agonists or antagonists, orexin receptorantagonists, urocortin binding protein antagonists, glucagon-likepeptide-1 receptor agonists, ciliary neutrotrophic factors (such asAxokine™ available from Regeneron Pharmaceuticals, Inc., Tarrytown, N.Y.and Procter & Gamble Company, Cincinnati, Ohio), human agouti-relatedproteins (AGRP), ghrelin receptor antagonists, histamine 3 receptorantagonists or reverse agonists, neuromedin U receptor agonists,noradrenergic anorectic agents (for example, phentemine, mazindol andthe like) and appetite suppressants (for example, bupropion). In someembodiments, the anti-obesity agent is selected from the groupconsisting of orlistat, sibutrine, bromocriptine, ephedrine, leptin, andpseudoephedrine.

In a combination therapy according to the present invention, the BRS-3agonist according to the present invention and the DPP-IV inhibitoraccording to the present invention can be administered simultaneously orat separate intervals. When administered simultaneously the BRS-3agonist and the DPP-IV inhibitor can be incorporated into a singlepharmaceutical composition or into separate compositions, e.g., theBRS-3 agonist in one composition and the DPP-IV inhibitor in anothercomposition. Each of these compositions may be formulated with commonexcipients, diluents or carriers, and compressed into tablets, orformulated elixirs or solutions; and as sustained relief dosage formsand the like. The BRS-3 agonist and DPP-IV inhibitor may be administeredvia different routes. For example, the BRS-3 agonist may be administeredorally via tablet and the DPP-IV inhibitor may be administered viainhalation.

When separately administered, therapeutically effective amounts of theBRS-3 agonist and the DPP-IV inhibitor according to the presentinvention are administered on a different schedule. One may beadministered before the other as long as the time between the twoadministrations falls within a therapeutically effective interval. Atherapeutically effective interval is a period of time beginning whenone of either (a) the BRS-3 agonist or (b) the DPP-IV inhibitor isadministered to a mammal and ending at the limit of the beneficialeffect in the treatment of the combination of (a) and (b).

In one aspect, the present invention features a pharmaceuticalcomposition comprising or consisting essentially of a combination of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention,together with at least one pharmaceutically acceptable carrier.

In one aspect, the present invention features a pharmaceuticalcomposition comprising or consisting essentially of a combination of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention,together with at least one pharmaceutically acceptable carrier. Thepresent invention also relates to a dosage form of the pharmaceuticalcomposition wherein the BRS-3 agonist and the DPP-IV inhibitor are inamounts sufficient to give an effect in lowering a blood glucose levelin a subject. In certain embodiments, the blood glucose level is anelevated blood glucose level.

In one aspect, the present invention features a pharmaceuticalcomposition comprising or consisting essentially of a combination of anamount of a BRS-/3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention,together with at least one pharmaceutically acceptable carrier. Thepresent invention also relates to a dosage form of the pharmaceuticalcomposition wherein the BRS-3 agonist and the DPP-IV inhibitor are inamounts sufficient to give an effect in lowering a blood glucose levelin a subject, and wherein the amount of the BRS-3 agonist alone and theamount of the DPP-IV inhibitor alone are therapeutically ineffective inlowering the blood glucose level in the subject. In certain embodiments,the blood glucose level is an elevated blood glucose level.

In one aspect, the present invention features a pharmaceuticalcomposition comprising or consisting essentially of a combination of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention,together with at least one pharmaceutically acceptable carrier. Thepresent invention also relates to a dosage form of the pharmaceuticalcomposition wherein the BRS-3 agonist and the DPP-IV inhibitor are inamounts sufficient to give an effect in lowering a blood glucose levelin a subject, and wherein the effect is a synergistic effect. In certainembodiments, the blood glucose level is an elevated blood glucose level.

In one aspect, the present invention relates to a pharmaceuticalcomposition comprising or consisting essentially of a combination of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention,together with at least one pharmaceutically acceptable carrier. Thepresent invention also relates to a dosage form of the pharmaceuticalcomposition wherein the BRS-3 agonist and the DPP-IV inhibitor are inamounts sufficient to give an effect in lowering a blood glucose levelin a subject, wherein the effect is a synergistic effect, and whereinthe amount of the BRS-3 agonist alone and the amount of the DPP-IVinhibitor alone are therapeutically ineffective in lowering the bloodglucose level in the subject. In certain embodiments, the blood glucoselevel is an elevated blood glucose level.

In one aspect, the present invention relates to a pharmaceuticalcomposition comprising or consisting essentially of a combination of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention,together with at least one pharmaceutically acceptable carrier. Thepresent invention also relates to a dosage form of the pharmaceuticalcomposition wherein the BRS-3 agonist and the DPP-IV inhibitor are inamounts sufficient to give an effect in lowering a blood glucose levelin a subject, wherein the effect given by the combination of the amountof the BRS-3 agonist and the amount of the DPP-IV inhibitor is greaterthan the effect given by the amount of the BRS-3 agonist alone and theeffect given by the amount of the DPP-IV inhibitor alone. In certainembodiments, the blood glucose level is an elevated blood glucose level.

In one aspect, the present invention features a pharmaceuticalcomposition comprising or consisting essentially of a combination of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention,together with at least one pharmaceutically acceptable carrier. Thepresent invention also relates to a dosage form of the pharmaceuticalcomposition wherein the BRS-3 agonist and the DPP-IV inhibitor are inamounts sufficient to give an effect in increasing a blood GLP-1 levelin a subject.

In one aspect, the present invention features a pharmaceuticalcomposition comprising or consisting essentially of a combination of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention,together with at least one pharmaceutically acceptable carrier. Thepresent invention also relates to a dosage form of the pharmaceuticalcomposition wherein the BRS-3 agonist and the DPP-IV inhibitor are inamounts sufficient to give an effect in increasing a blood GLP-1 levelin a subject, and wherein the amount of the BRS-3 agonist alone and theamount of the DPP-IV inhibitor alone are therapeutically ineffective inincreasing a blood GLP-1 level in the subject.

In one aspect, the present invention features a pharmaceuticalcomposition comprising or consisting essentially of a combination of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention,together with at least one pharmaceutically acceptable carrier. Thepresent invention also relates to a dosage form of the pharmaceuticalcomposition wherein the BRS-3 agonist and the DPP-IV inhibitor are inamounts sufficient to give an effect in increasing a blood GLP-1 levelin a subject, and wherein the effect is a synergistic effect.

In one aspect, the present invention relates to a pharmaceuticalcomposition comprising or consisting essentially of a combination of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention,together with at least one pharmaceutically acceptable carrier. Thepresent invention also relates to a dosage form of the pharmaceuticalcomposition wherein the BRS-3 agonist and the DPP-IV inhibitor are inamounts sufficient to give an effect in increasing a blood GLP-1 levelin a subject, wherein the effect is a synergistic effect, and whereinthe amount of the BRS-3 agonist alone and the amount of the DPP-IVinhibitor alone are therapeutically ineffective in increasing a bloodGLP-1 level in the subject.

In one aspect, the present invention relates to a pharmaceuticalcomposition comprising or consisting essentially of a combination of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention,together with at least one pharmaceutically acceptable carrier. Thepresent invention also relates to a dosage form of the pharmaceuticalcomposition wherein the BRS-3 agonist and the DPP-IV inhibitor are inamounts sufficient to give an effect in increasing a blood GLP-1 levelin a subject, wherein the effect given by the combination of the amountof the BRS-3 agonist and the amount of the DPP-IV inhibitor is greaterthan the effect given by the amount of the BRS-3 agonist alone and theeffect given by the amount of the DPP-IV inhibitor alone.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in anamount to achieve their intended purpose. In some embodiments, apharmaceutical composition of the present invention is understood to beuseful for treating or preventing diabetes and conditions relatedthereto. Diabetes and conditions related thereto are according to thepresent invention. In some embodiments, a pharmaceutical composition ofthe present invention is understood to be useful for treating orpreventing a condition ameliorated by increasing a blood GLP-1 level.Conditions ameliorated by increasing a blood GLP-1 level are accordingto the present invention.

In certain embodiments of the combination therapy of the presentinvention, the amount of BRS-3 agonist according to the presentinvention and the amount of DPP-IV inhibitor according to the presentinvention are provided in amounts to give a synergistic effect inlowering a blood glucose level in a subject. In certain embodiments, theblood glucose level is an elevated blood glucose level. Determination ofthe amounts of BRS-3 agonist and DPP-IV inhibitor providing asynergistic effect in lowering blood glucose level in a subject is wellwithin the capability of those skilled in the art, especially in lightof the detailed disclosure provided herein. In one embodiment of thecombination therapy of the present invention, the amount of BRS-3agonist according to the present invention and the amount of DPP-IVinhibitor according to the present invention are provided in amounts togive a synergistic effect in lowering a blood glucose level in asubject, wherein the amount of the BRS-3 agonist alone and the amount ofthe DPP-IV inhibitor alone are therapeutically ineffective in loweringthe blood glucose level in the subject. In certain embodiments, theblood glucose level is an elevated blood glucose level. Determination ofthe amounts of BRS-3 agonist and DPP-IV inhibitor providing asynergistic effect in lowering blood glucose level in a subject, whereinthe amount of the BRS-3 agonist alone and the amount of the DPP-IVinhibitor alone are therapeutically ineffective in lowering bloodglucose level in the subject, is well within the capability of thoseskilled in the art, especially in light of the detailed disclosureprovided herein.

In certain embodiments of the combination therapy of the presentinvention, the amount of BRS-3 agonist according to the presentinvention and the amount of DPP-IV inhibitor according to the presentinvention are provided in amounts to give a synergistic effect inincreasing a blood GLP-1 level in a subject. Determination of theamounts of BRS-3 agonist and DPP-IV inhibitor providing a synergisticeffect in increasing a blood GLP-1 level in a subject is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein. In one embodiment of thecombination therapy of the present invention, the amount of BRS-3agonist according to the present invention and the amount of DPP-IVinhibitor according to the present invention are provided in amounts togive a synergistic effect in increasing a blood GLP-1 level in asubject, wherein the amount of the BRS-3 agonist alone and the amount ofthe DPP-IV inhibitor alone are therapeutically ineffective in increasinga blood GLP-1 level in the subject. Determination of the amounts ofBRS-3 agonist and DPP-IV inhibitor providing a synergistic effect inincreasing a blood GLP-1 level in a subject, wherein the amount of theBRS-3 agonist alone and the amount of the DPP-IV inhibitor alone aretherapeutically ineffective in increasing a blood GLP-1 level in thesubject, is well within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein.

The data obtained from animal studies, including but not limited tostudies using mice, rats, rabbits, pigs, and non-human primates, can beused in formulating a range of dosage for use in humans. In general, oneskilled in the art uiderstands how to extrapolate in vivo data obtainedin an animal model system to another, such as a human. In somecircumstances, these extrapolations may merely be based on the weight ofthe animal model in comparison to another, such as a human; in othercircumstances, these extrapolations are not simply based on weights butrather incorporate a variety of factors. Representative factors includethe type, age, weight, sex, diet and medical condition of the patient,the severity of the disease, the route of administration,pharmacological considerations such as the activity, efficacy,pharmacokinetic and toxicology profiles of the particular compoundemployed, whether a drug delivery system is utilized, on whether anacute or chronic disease state is being treated or prophylaxis isconducted or on whether further active compounds are administered inaddition to the compounds of the present invention and as part of a drugcombination. The dosage regimen for treating a disease condition withthe compounds and/or compositions of this invention is selected inaccordance with a variety factors as cited above. Thus, the actualdosage regimen employed may vary widely and therefore may deviate from apreferred dosage regimen and one skilled in the art will recognize thatdosage and dosage regimen outside these typical ranges can be testedand, where appropriate, may be used in the methods of this invention.

An exemplary and preferred animal model system is oral glucose tolerancetest (oGTT) in mice (see, Example 1). In this model, by way ofillustration and not limitation, an amount of a BRS-3 agonist alone or aDPP-IV inhibitor alone which is therapeutically ineffective is an amountof the BRS-3 agonist alone or the DPP-IV inhibitor alone producing anArea Under Curve (AUC) inhibition of glycemic excursion less than orequal to about 30%, less than about 25%, less than about 20%, less thanabout 15%, less than about 10%, or less than about 5%, more preferablyless than about 25%, less than about 20%, less than about 15%, less thanabout 10%, or less than about 5%. In this model, by way of illustrationand not limitation, an amount of a BRS-3 agonist alone or a DPP-IVinhibitor alone which is therapeutically ineffective is an amount of theBRS-3 agonist alone or the DPP-IV inhibitor alone producing an AreaUnder Curve (AUC) inhibition of glycemic excursion about 0-30%, about0-25%, about 0-20%, about 0-15%, about 0-10%, or about 0-5%, morepreferably about 0-25%, about 0-20%, about 0-15%, about 0-10%, or about0-5%. In this model, by way of illustration and not limitation, atherapeutically effective amount of a combination of a BRS-3 agonist anda DPP-IV inhibitor in accordance with the present invention is an amountof the combination producing an Area Under Curve (AUC) inhibition ofglycemic excursion greater than about 30%, greater than about 35%,greater than about 40%, greater than about 45%, greater than about 50%,greater than about 55%, greater than about 60%, greater than about 65%,greater than about 70%, greater than about 75%, greater than about 80%,greater than about 85%, greater than about 90%, or greater than about95%, more preferably greater than about 35%, greater than about 40%,greater than about 45%, greater than about 50%, greater than about 55%,greater than about 60%, greater than about 65%, greater than about 70%,or greater than about 75%, greater than about 80%, greater than about85%, greater than about 90%, or greater than about 95%.

Dosage amount and interval may be adjusted in order to provide asynergistic effect in lowering a blood glucose level in the subject inaccordance with the present invention or to provide a synergistic effectin increasing a blood GLP-1 level in the subject in accordance with thepresent invention. In certain embodiments, the blood glucose level is anelevated blood glucose level. It will be appreciated that the exactdosage of a BRS-3 agonist or DPP-IV inhibitor in accordance with thepresent invention will vary depending on the combination of the BRS-3agonist and DPP-IV inhibitor, its potency, the mode of administration,the age and weight of the patient and the severity of the condition tobe treated. The exact formulation, route of administration and dosagecan be chosen by the individual physician in view of the patient'scondition. By way of illustration and not limitation, an amount of BRS-3agonist or DPP-IV inhibitor providing a synergistic effect in lowering ablood glucose level in the subject in accordance with the presentinvention or providing a synergistic effect in increasing a blood GLP-1level in the subject in accordance with the present invention is lessthan about 0.001 mg/kg body weight, less than about 0.005 mg/kg bodyweight, less than about 0.01 mg/kg body weight, less than about 0.05mg/kg body weight, less than about 0.1 mg/kg body weight, less thanabout 0.5 mg/kg body weight, less than about 1 mg/kg body weight, lessthan about 5 mg/kg body weight, less than about 10 mg/kg body weight,less than about 50 mg/kg body weight, or less than about 100 mg/kg bodyweight. In certain embodiments, the blood glucose level is an elevatedblood glucose level. In some embodiments, an amount of BRS-3 agonist orDPP-IV inhibitor providing a synergistic effect in lowering a bloodglucose level in the subject in accordance with the present invention orproviding a synergistic effect in increasing a blood GLP-1 level in thesubject in accordance with the present invention is less than about0.001-100 mg/kg body weight, less than about 0.001-50 mg/kg body weight,less than about 0.001-10 mg/kg body weight, less than about 0.001-5mg/kg body weight, less than about 0.001-1 mg/kg body weight, less thanabout 0.001 to 0.5 mg/kg body weight, less than about 0.001-0.1 mg/kgbody weight, less than about 0.001-0.05 mg/kg body weight, less thanabout 0.001-0.01 mg/kg body weight, or less than about 0.001-0.005 mg/kgbody weight. In certain embodiments, the blood glucose level is anelevated blood glucose level. In some embodiments, an amount of BRS-3agonist or DPP-IV inhibitor providing a synergistic effect in lowering ablood glucose level in the subject in accordance with the presentinvention or providing a synergistic effect in increasing a blood GLP-1level in the subject in accordance with the present invention is about0.001-100 mg/kg body weight, about 0.001-50 mg/kg body weight, about0.001-10 mg/kg body weight, about 0.001-5 mg/kg body weight, about 0.001to 1 mg/kg body weight, about 0.001-0.5 mg/kg body weight, about0.001-0.1 mg/kg body weight, about 0.001-0.05 mg/kg body weight, about0.001-0.01 mg/kg body weight, or about 0.001-0.005 mg/kg body weight. Incertain embodiments, the blood glucose level is an elevated bloodglucose level.

An additional exemplary and preferred animal model system is increase ofa blood GLP-1 level after glucose challenge in mice (see, Example 15).

Dosage amount and interval may be adjusted individually to provideplasma levels of BRS-3 agonist according to the present invention andDPP-IV inhibitor according to the present invention which provide asynergistic effect in lowering a blood glucose level in the subjectaccording to the present invention or provide a synergistic effect inincreasing a blood GLP-1 level in the subject according to the presentinvention. In certain embodiments, the blood glucose level is anelevated blood glucose level. Dosage intervals can also be determinedusing the value for a selected range of BRS-3 agonist concentration orthe value for a selected range of DPP-IV inhibitor concentrationproviding a synergistic effect in lowering a blood glucose level in thesubject according to the present invention or providing a synergisticeffect in increasing a blood GLP-1 level in the subject according to thepresent invention. In certain embodiments, the blood glucose level is anelevated blood glucose level. BRS-3 agonist and DPP-IV inhibitor shouldbe administered using a regimen that maintains plasma levels within theselected range of BRS-3 agonist concentration and DPP-IV inhibitorconcentration, respectively, for 10-90% of the time, preferably between30-99% of the time, and most preferably between 50-90% of the time. Incases of local administration or selective uptake, the range of BRS-3agonist concentration or the range of DPP-IV inhibitor concentrationproviding a synergistic effect in lowering a blood glucose level in thesubject according to the present invention or providing a synergisticeffect in increasing a blood GLP-1 level in the subject according to thepresent invention may not be related to plasma concentration. In certainembodiments, the blood glucose level is an elevated blood glucose level.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration, and the judgement of theprescribing physician.

In one aspect, the present invention accordingly features a method oftreating or preventing diabetes or a condition related theretocomprising administering to a subject in need thereof a therapeuticallyeffective amount of a composition comprising or consisting essentiallyof an amount of a BRS-3 agonist according to the present invention andan amount of a DPP-IV inhibitor according to the present invention.

In one aspect, the present invention relates to a method of treating orpreventing diabetes or a condition related thereto comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising or consisting essentially of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention. In arelated aspect, the present invention features said method wherein theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to give an effect in lowering a blood glucose level in thesubject. In certain embodiments, the blood glucose level is an elevatedblood glucose level.

In one aspect, the present invention relates to a method of treating orpreventing diabetes or a condition related thereto comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising or consisting essentially of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention. In arelated aspect, the present invention features said mehod wherein theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to give an effect in lowering a blood glucose level in thesubject, and wherein the amount of the BRS-3 agonist alone and theamount of the DPP-IV inhibitor alone are therapeutically ineffective inlowering the blood glucose level in the subject. In certain embodiments,the blood glucose level is an elevated blood glucose level.

In one aspect, the present invention relates to a method of treating orpreventing diabetes or a condition related thereto comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising or consisting essentially of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention. In arelated aspect, the present invention features said method wherein theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to give an effect in lowering a blood glucose level in thesubject, and wherein the effect is a synergistic effect. In certainembodiments, the blood glucose level is an elevated blood glucose level.

In one aspect, the present invention relates to a method of treating orpreventing diabetes or a condition related thereto comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising or consisting essentially of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention. In arelated aspect, the present invention features said method wherein theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to give an effect in lowering a blood glucose level in thesubject wherein the effect is a synergistic effect, and wherein theamount of the BRS-3 agonist alone and the amount of the DPP-IV inhibitoralone are therapeutically ineffective in lowering the blood glucoselevel in the subject. In certain embodiments, the blood glucose level isan elevated blood glucose level.

In one aspect, the present invention relates to a method of treating orpreventing diabetes or a condition related thereto comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising or consisting essentially of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention. In arelated aspect, the present invention features said method wherein theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to give an effect in lowering a blood glucose level in thesubject, wherein the effect given by the combination of the amount ofthe BRS-3 agonist and the amount of the DPP-IV inhibitor is greater thanthe effect given by the amount of the BRS-3 agonist alone and the effectgiven by the amount of the DPP-IV inhibitor alone. In certainembodiments, the blood glucose level is an elevated blood glucose level.

A combination therapy of the present invention is useful in treating orpreventing diabetes or a condition related thereto in a mammal,including and most preferably in a human. In some embodiments, diabetesis Type 1 diabetes. In some preferred embodiments, diabetes is Type 2diabetes. A condition related to diabetes includes, but is not limitedto, hyperglycemia, impaired glucose tolerance, insulin resistance,pancreatic beta-cell insufficiency, enteroendocrine cell insufficiency,glucosuria, metabolic acidosis, cataracts, diabetic nephropathy,diabetic neuropathy, diabetic retinopathy, diabetic coronary arterydisease, diabetic cerebrovascular disease, diabetic peripheral vasculardisease, metabolic syndrome, hyperlipidemia, atherosclerosis, stroke,hypertension, and obesity. It is understood that conditions related todiabetes can be included in embodiments individually or in anycombination.

In one aspect, the present invention accordingly features a method ofreducing body mass comprising administering to a subject in need thereofa therapeutically effective amount of a composition comprising orconsisting essentially of an amount of a BRS-3 agonist according to thepresent invention and an amount of a DPP-IV inhibitor according to thepresent invention.

In one aspect, the present invention relates to a method of reducingbody mass comprising administering to a subject in need thereof atherapeutically effective amount of a composition comprising orconsisting essentially of an amount of a BRS-3 agonist according to thepresent invention and an amount of a DPP-IV inhibitor according to thepresent invention. In a related aspect, the present invention featuressaid method wherein the BRS-3 agonist and the DPP-IV inhibitor areadministered in amounts sufficient to give an effect in increasing ablood GLP-1 level in the subject.

In one aspect, the present invention relates to a method of reducingbody mass comprising administering to a subject in need thereof atherapeutically effective amount of a composition comprising orconsisting essentially of an amount of a BRS-3 agonist according to thepresent invention and an amount of a DPP-IV inhibitor according to thepresent invention. In a related aspect, the present invention featuressaid method wherein the BRS-3 agonist and the DPP-IV inhibitor areadministered in amounts sufficient to give an effect in increasing ablood GLP-1 level in the subject, and wherein the amount of the BRS-3agonist alone and the amount of the DPP-IV inhibitor alone aretherapeutically ineffective in increasing a blood GLP-1 level in thesubject.

In one aspect, the present invention relates to a method of reducingbody mass comprising administering to a subject in need thereof atherapeutically effective amount of a composition comprising orconsisting essentially of an amount of a BRS-3 agonist according to thepresent invention and an amount of a DPP-IV inhibitor according to thepresent invention. In a related aspect, the present invention featuressaid method wherein the BRS-3 agonist and the DPP-IV inhibitor areadministered in amounts sufficient to give an effect in increasing ablood GLP-1 level in the subject, and wherein the effect is asynergistic effect.

In one aspect, the present invention relates to a method of reducingbody mass comprising administering to a subject in need thereof atherapeutically effective amount of a composition comprising orconsisting essentially of an amount of a BRS-3 agonist according to thepresent invention and an amount of a DPP-IV inhibitor according to thepresent invention. In a related aspect, the present invention featuressaid method wherein the BRS-3 agonist and the DPP-IV inhibitor areadministered in amounts sufficient to give an effect in increasing ablood GLP-1 level in the subject, wherein the effect is a synergisticeffect, and wherein the amount of the BRS-3 agonist alone and the amountof the DPP-IV inhibitor alone are therapeutically ineffective inincreasing a blood GLP-1 level in the subject.

In one aspect, the present invention relates to a method of reducingbody mass comprising administering to a subject in need thereof atherapeutically effective amount of a composition comprising orconsisting essentially of an amount of a BRS-3 agonist according to thepresent invention and an amount of a DPP-IV inhibitor according to thepresent invention. In a related aspect, the present invention featuressaid method wherein the BRS-3 agonist and the DPP-IV inhibitor areadministered in amounts sufficient to give an effect in increasing ablood GLP-1 level in the subject, wherein the effect given by thecombination of the amount of the BRS-3 agonist and the amount of theDPP-IV inhibitor is greater than the effect given by the amount of theBRS-3 agonist alone and the effect given by the amount of the DPP-IVinhibitor alone.

In one aspect, the present invention accordingly features a method oftreating or preventing obesity or a condition related thereto comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising or consisting essentially of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention.

In one aspect, the present invention relates to a method of treating orpreventing obesity or a condition related thereto comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising or consisting essentially of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention. In arelated aspect, the present invention features said method wherein theDRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to give an effect in increasing a blood GLP-1 level in thesubject.

In one aspect, the present invention relates to a method of treating orpreventing obesity or a condition related thereto comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising or consisting essentially of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention. In arelated aspect, the present invention features said method wherein theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to give an effect in increasing a blood GLP-1 level in thesubject, and wherein the amount of the BRS-3 agonist alone and theamount of the DPP-IV inhibitor alone are therapeutically ineffective inincreasing a blood GLP-1 level in the subject.

In one aspect, the present invention relates to a method of treating orpreventing obesity or a condition related thereto comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising or consisting essentially of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention. In arelated aspect, the present invention features said method wherein theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to give an effect in increasing a blood GLP-1 level in thesubject, and wherein the effect is a synergistic effect.

In one aspect, the present invention relates to a method of treating orpreventing obesity or a condition related thereto comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising or consisting essentially of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention. In arelated aspect, the present invention features said method wherein theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to give an effect in increasing a blood GLP-1 level in thesubject, wherein the effect is a synergistic effect, and wherein theamount of the BRS-3 agonist alone and the amount of the DPP-IV inhibitoralone are therapeutically ineffective in increasing a blood GLP-1 levelin the subject.

In one aspect, the present invention relates to a method of treating orpreventing obesity or a condition related thereto comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising or consisting essentially of anamount of a BRS-3 agonist according to the present invention and anamount of a DPP-IV inhibitor according to the present invention. In arelated aspect, the present invention features said method wherein theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to give an effect in increasing a blood GLP-1 level in thesubject, wherein the effect given by the combination of the amount ofthe BRS-3 agonist and the amount of the DPP-IV inhibitor is greater thanthe effect given by the amount of the BRS-3 agonist alone and the effectgiven by the amount of the DPP-IV inhibitor alone.

A combination therapy of the present invention is useful in treating orpreventing obesity or a condition related thereto in a mammal, includingand most preferably in a human. A condition related to obesity includes,but is not limited to, hypertension, congestive cardiomyopathy,varicosities, pulmonary embolism, coronary heart disease, stroke,idiopathic intracranial hypertension, meralgia parethetica, dyspnea,obstructive sleep apnea, hypoventilation syndrome, Pickwickian syndrome,asthma, immobility, degenerative osteoartritis, low back pain, striaedistensae or “stretch marks,” venous stasis of the lower extremities,lymphedema, cellulitis, intertrigo, carbuncles, acanthosis nigricans,skin tags, gastro-esophageal reflux disorder, nonalcoholic fattyliver/steatohepatitis, cholelithiasis, hernias, colon cancer, stressincontinence, obesity-related glomerulopathy, breast and uterine cancer,depression and low self-esteem, impaired quality of life, metabolicsyndrome, Type 2 diabetes, dyslipidemia, hyperandrogenemia in women,polycystic ovarian syndrome, dysmenorrhea, infertility, pregnancycomplications, and male hypogonadism. In certain embodiments, thecondition related to obesity is selected from the group consisting ofhypertension, congestive cardiomyopathy, coronary heart disease, stroke,dyslipidemia, metabolic syndrome, and Type 2 diabetes. It is understoodthat conditions related to obesity can be included in embodimentsindividually or in any combination.

In one aspect, the present invention accordingly features a method oftreating or preventing a condition ameliorated by increasing a bloodGLP-1 level comprising administering to a subject in need thereof atherapeutically effective amount of a composition comprising orconsisting essentially of an amount of a BRS-3 agonist according to thepresent invention and an amount of a DPP-IV inhibitor according to thepresent invention.

In one aspect, the present invention relates to a method of treating orpreventing a condition ameliorated by increasing a blood GLP-1 levelcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a composition comprising or consisting essentiallyof an amount of a BRS-3 agonist according to the present invention andan amount of a DPP-IV inhibitor according to the present invention. In arelated aspect, the present invention features said method wherein theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to give an effect in increasing a blood GLP-1 level in thesubject.

In one aspect, the present invention relates to a method of treating orpreventing a condition ameliorated by increasing a blood GLP-1 levelcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a composition comprising or consisting essentiallyof an amount of a BRS-3 agonist according to the present invention andan amount of a DPP-IV inhibitor according to the present invention. In arelated aspect, the present invention features said method wherein theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to give an effect in increasing a blood GLP-1 level in thesubject, and wherein the amount of the BRS-3 agonist alone and theamount of the DPP-IV inhibitor alone are therapeutically ineffective inincreasing a blood GLP-1 level in the subject.

In one aspect, the present invention relates to a method of treating orpreventing a condition ameliorated by increasing a blood GLP-1 levelcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a composition comprising or consisting essentiallyof an amount of a BRS-3 agonist according to the present invention andan amount of a DPP-IV inhibitor according to the present invention. In arelated aspect, the present invention features said method wherein theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to give an effect in increasing a blood GLP-1 level in thesubject, and wherein the effect is a synergistic effect.

In one aspect, the present invention relates to a method of treating orpreventing a condition ameliorated by increasing a blood GLP-1 levelcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a composition comprising or consisting essentiallyof an amount of a BRS-3 agonist according to the present invention andan amount of a DPP-IV inhibitor according to the present invention. In arelated aspect, the present invention features said method wherein theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to give an effect in increasing a blood GLP-1 level in thesubject, wherein the effect is a synergistic effect, and wherein theamount of the BRS-3 agonist alone and the amount of the DPP-IV inhibitoralone are therapeutically ineffective in increasing a blood GLP-1 levelin the subject.

In one aspect, the present invention relates to a method of treating orpreventing a condition ameliorated by increasing a blood GLP-1 levelcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a composition comprising or consisting essentiallyof an amount of a BRS-3 agonist according to the present invention andan amount of a DPP-IV inhibitor according to the present invention. In arelated aspect, the present invention features said method wherein theBRS-3 agonist and the DPP-IV inhibitor are administered in amountssufficient to give an effect in increasing a blood GLP-1 level in thesubject, wherein the effect given by the combination of the amount ofthe BRS-3 agonist and the amount of the DPP-IV inhibitor is greater thanthe effect given by the amount of the BRS-3 agonist alone and the effectgiven by the amount of the DPP-IV inhibitor alone.

A combination therapy of the present invention is useful in treating orpreventing a condition ameliorated by increasing a blood GLP-1 level ina mammal, including and most preferably in a human. A conditionameliorated by increasing a blood GLP-1 level includes, but is notlimited to, diabetes, a condition related to diabetes, myocardialinfarction, learning impairment, memory impairment, and aneurodegenerative disorder, wherein a condition related to diabetesincludes, but is not limited to, hyperglycemia, impaired glucosetolerance, insulin resistance, pancreatic beta-cell insufficiency,enteroendocrine cell insufficiency, glucosuria, metabolic acidosis,cataracts, diabetic nephropathy, diabetic neuropathy, diabeticretinopathy, diabetic coronary artery disease, diabetic cerebrovasculardisease, diabetic peripheral vascular disease, metabolic syndrome,hyperlipidernia, atherosclerosis, stroke, hypertension, and obesity,wherein a neurodegenerative disorder includes, but is not limited to,excitotoxic brain damage caused by severe epileptic seizures,Alzheimer's disease, Parkinson's disease, Huntington's disease,prion-associated disease, motor-neuron disease, traumatic brain injury,spinal cord injury, and peripheral neuropathy. In some embodiments,diabetes is Type 1 diabetes. In some preferred embodiments, diabetes isType 2 diabetes. It is understood that conditions ameliorated byincreasing a blood GLP-1 level can be included in embodimentsindividually or in any combination.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, practice the present invention toits fullest extent. The foregoing detailed description is given forclearness of understanding only, and no unnecessary limitation should beunderstood therefrom, as modifications within the scope of the inventionmay become apparent to those skilled in the art.

Recombinant DNA techniques relating to the subject matter of the presentinvention and well known to those of ordinary skill in the art can befound, e.g, in Sambrook et al., Molecular Cloning: A Laboratory Manual(2001) Cold Spring Harbor Laboratory; the disclosure of which is herebyincorporated by reference in its entirety.

Throughout this application, various publications, patents and patentapplications are cited. The disclosures of these publications, patentsand patent applications referenced in this application are hereinincorporated by reference in their entirety into the present disclosure.Citation herein by Applicant of a publication, patent, or patentapplication is not an admission by Applicant of said publication,patent, or patent application as prior art.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, practice the present invention toits fullest extent. The following detailed examples are to be construedas merely illustrative, and not limitations of the preceding disclosurein any way whatsoever. Those skilled in the art will promptly recognizeappropriate variations from the procedures.

Example 1 Full-Length Cloning of Endogenous Human BRS-3

Polynucleotide encoding endogenous human BRS-3 was cloned by RT-PCRusing the BRS-3 specific primers 5′-ACAGAATTCAGAAGAAATGGCTCAAAGGCA-3′(SEQ ID NO:3; sense with EcoRI site, ATG as initiation codon) and5′-CATGGATCCT7GAAAAGCTAGAATCTGTCC-3′ (SEQ ID NO:4; antisense with BamHIsite, CTA as antisense of stop codon)

and human uterus cDNA (Clontech) as template. TaqPlus Precision™ DNApolymerase (Stratagene) was used for amplification by the followingcycle with step 2 to step 4 repeated 25 times:

94° C., 3 minutes; 94° C., 1 minute; 56° C., 1 minute; 72° C., 1 minute20 sec; 72° C., 10 minutes.

A 1.23 Kb PCR fragment of predicted size was isolated, digested withEcoRI and BamHI, and cloned into the pCMV expression vector andsequenced using the T7 DNA sequenase kit (Amersham). See, SEQ ID NO:1for nucleic acid sequence and SEQ ID NO:2 for the deduced amino acidsequence.

Example 2 Receptor Expression

Although a variety of cells are available to the art for the expressionof G protein-coupled receptors, it is most preferred that mammaliancells or melanophores be utilized. The following are illustrative; thoseof ordinary skill in the art are credited with the ability to determinethose techniques that are preferentially beneficial for the needs of theartisan. See, e.g., Example 6, infra, as it relates to melanophores.

a. Transient Transfection

On day one, 6×10⁶/10 cm dish of 293 cells are plated out. On day two,two reaction tubes are prepared (the proportions to follow for each tubeare per plate): tube A is prepared by mixing 4 μg DNA (e.g., pCMVvector; pCMV vector with receptor cDNA, etc.) in 0.5 ml serum free DMEM(Gibco BRL); tube B is prepared by mixing 24 μl lipofectamine (GibcoBRL) in 0.5 ml serum free DMEM. Tubes A and B are admixed by inversions(several times), followed by incubation at room temperature for 30-45min. The admixture is referred to as the “transfection mixture”. Plated293 cells are washed with 1×PBS, followed by addition of 5 ml serum freeDMEM. 1 ml of the transfection mixture is added to the cells, followedby incubation for 4 hrs at 37° C./5% CO₂. The transfection mixture isremoved by aspiration, followed by the addition of 10 ml of DMEM/10%Fetal Bovine Serum. Cells are incubated at 37° C./5% CO₂. After 48 hrincubation, cells are harvested and utilized for analysis.

b. Stable Cell Lines

Approximately 12×10⁶ 293 cells are plated on a 15 cm tissue cultureplate. Grown in DME High Glucose Medium containing ten percent fetalbovine serum and one percent sodium pyruvate, L-glutamine, andantibiotics. Twenty-four hours following plating of 293 cells (or to˜80% confluency), the cells are transfected using 12 μg of DNA (e.g.,pCMV vector with receptor cDNA). The 12 μg of DNA is combined with 60 μlof lipofectamine and 2 ml of DME High Glucose Medium without serum. Themedium is aspirated from the plates and the cells are washed once withmedium without serum. The DNA, lipofectamine, and medium mixture areadded to the plate along with 10 ml of medium without serum. Followingincubation at 37° C. for four to five hours, the medium is aspirated and25 ml of medium containing serum is added. Twenty-four hours followingtransfection, the medium is aspirated again, and fresh medium with serumis added. Forty-eight hours following transfection, the medium isaspirated and medium with serum is added containing geneticin (G418drug) at a final concentration of approximately 12×10⁶ 293 cells areplated on a 15 cm tissue culture plate. Grown in DME High Glucose Mediumcontaining ten percent fetal bovine serum and one percent sodiumpyruvate, L-glutamine, and antibiotics. Twenty-four hours followingplating of 293 cells (or to ˜80% confluency), the cells are transfectedusing 12 μg of DNA (e.g., pCMV vector with receptor cDNA). The 12 μg ofDNA is combined with 60 μl of lipofectamine and 2 ml of DME High GlucoseMedium without serum. The medium is aspirated from the plates and thecells are washed once with medium without serum. The DNA, lipofectamine,and medium mixture are added to the plate along with 10 ml of mediumwithout serum. Following incubation at 37° C. for four to five hours,the medium is aspirated and 25 ml of medium containing serum is added.Twenty-four hours following transfection, the medium is aspirated again,and fresh medium with serum is added. Forty-eight hours followingtransfection, the medium is aspirated and medium with serum is addedcontaining geneticin (G418 drug) at a final concentration of 500 μg/ml.The transfected cells now undergo selection for positively transfectedcells containing the G418 resistance gene. The medium is replaced everyfour to five days as selection occurs. During selection, cells are grownto create stable pools, or split for stable clonal selection.

Example 3 Assays for Screening Candidate Compounds as BRS-3 Agonists

A variety of approaches are available for screening candidate compoundsas BRS-3 agonists. The following are illustrative; those of ordinaryskill in the art are credited with the ability to determine thosetechniques that are preferentially beneficial for the needs of theartisan. Assays for screening compounds as agonists of a Gprotein-coupled receptor are well known to the skilled artisan (see,e.g., International Application WO 02/42461).

1. Membrane Binding Assays: [³⁵S]GTPγS Assay

When a G protein-coupled receptor is in its active state, either as aresult of ligand binding or constitutive activation, the receptorcouples to a G protein and stimulates the release of GDP and subsequentbinding of GTP to the G protein. The alpha subunit of the Gprotein-receptor complex acts as a GTPase and slowly hydrolyzes the GTPto GDP, at which point the receptor normally is deactivated. Activatedreceptors continue to exchange GDP for GTP. The non-hydrolyzable GTPanalog, [³⁵S]GTPγS, can be utilized to demonstrate enhanced binding of[³⁵S]GTPγS to membranes expressing activated receptors. The advantage ofusing [³⁵S]GTPγS binding to measure activation is that: (a) it isgenerically applicable to all G protein-coupled receptors; (b) it isproximal at the membrane surface making it less likely to pick-upmolecules which affect the intracellular cascade.

The assay utilizes the ability of G protein coupled receptors tostimulate [³⁵S]GTPγS binding to membranes expressing the relevantreceptors. The assay is generic and has application to drug discovery atall G protein-coupled receptors.

Membrane Preparation

In some embodiments, membranes comprising a G protein-coupled receptorof the invention and for use in the identification of candidatecompounds as, e.g., agonists of the receptor, are preferably prepared asfollows:

a. Materials

“Membrane Scrape Buffer” is comprised of 20 mM HEPES and 10 mM EDTA, pH7.4; “Membrane Wash Buffer” is comprised of 20 mM HEPES and 0.1 mM EDTA,pH 7.4; “Binding Buffer” is comprised of 20 mM BEPES, 100 mM NaCl, and10 mM MgCl₂, pH 7.4.

b. Procedure

All materials will be kept on ice throughout the procedure. Firstly, themedia will be aspirated from a confluent monolayer of cells, followed byrinse with 10 ml cold PBS, followed by aspiration. Thereafter, 5 ml ofMembrane Scrape Buffer will be added to scrape cells; this will befollowed by transfer of cellular extract into 50 ml centrifuge tubes(centrifuged at 20,000 rpm for 17 minutes at 4° C.). Thereafter, thesupernatant will be aspirated and the pellet will be resuspended in 30ml Membrane Wash Buffer followed by centrifuge at 20,000 rpm for 17minutes at 4° C. The supernatant will then be aspirated and the pelletresuspended in Binding Buffer. This will then be homogenized using aBrinkman Polytron™ homogenizer (15-20 second bursts until the allmaterial is in suspension). This is referred to herein as “MembraneProtein”.

Bradford Protein Assay

Following the homogenization, protein concentration of the membraneswill be determined using the Bradford Protein Assay (protein can bediluted to about 1.5 mg/ml, aliquoted and frozen (−80° C.) for lateruse; when frozen, protocol for use will be as follows: on the day of theassay, frozen Membrane Protein is thawed at room temperature, followedby vortex and then homogenized with a Polytron at about 12×1,000 rpm forabout 5-10 seconds; it is noted that for multiple preparations, thehomogenizer should be thoroughly cleaned between homogenization ofdifferent preparations).

a. Materials

Binding Buffer (as per above); Bradford Dye Reagent; Bradford ProteinStandard will be utilized, following manufacturer instructions (Biorad,cat. no. 500-0006).

b. Procedure

Duplicate tubes will be prepared, one including the membrane, and one asa control “blank”. Each contained 800 μl Binding Buffer. Thereafter, 10μl of Bradford Protein Standard (1 mg/ml) will be added to each tube,and 10 μl of membrane Protein will then be added to just one tube (notthe blank). Thereafter, 200 μl of Bradford Dye Reagent will be added toeach tube, followed by vortex of each. After five (5) minutes, the tubeswill be re-vortexed and the material therein will be transferred tocuvettes. The cuvettes will then be read using a CECIL 3041spectrophotometer, at wavelength 595.

Identification Assay

a. Materials

GDP Buffer consisted of 37.5 ml Binding Buffer and 2 mg GDP (Sigma, cat.no. G-7127), followed by a series of dilutions in Binding Buffer toobtain 0.2 μM GDP (final concentration of GDP in each well was 0.1 μMGDP); each well comprising a candidate compound, has a final volume of200 μl consisting of 100 μl GDP Buffer (final concentration, 0.1 μMGDP), 50 μl Membrane Protein in Binding Buffer, and 50 μl [³⁵S]GTPγS(0.6 nM) in Binding Buffer (2.5 μl [³⁵S]GTPγS per 10 ml Binding Buffer).

b. Procedure

Candidate compounds will be preferably screened using a 96-well plateformat (these can be frozen at −80° C.). Membrane Protein (or membraneswith expression vector excluding the Target GPCR, as control), will behomogenized briefly until in suspension. Protein concentration will thenbe determined using the Bradford Protein Assay set forth above. MembraneProtein (and control) will then be diluted to 0.25 mg/ml in BindingBuffer (final assay concentration, 12.5 μg/well). Thereafter, 100 μl GDPBuffer was added to each well of a Wallac Scintistrip™ (Wallac). A 5 ulpin-tool will then be used to transfer 5 μl of a candidate compound intosuch well (i.e., 5 μl in total assay volume of 200 μl is a 1:40 ratiosuch that the final screening concentration of the candidate compound is10 μM). Again, to avoid contamination, after each transfer step the pintool should be rinsed in three reservoirs comprising water (1×), ethanol(1×) and water (2×)—excess liquid should be shaken from the tool aftereach rinse and dried with paper and kimwipes. Thereafter, 50 μl ofMembrane Protein will be added to each well (a control well comprisingmembranes without the Target GPCR was also utilized), and pre-incubatedfor 5-10 minutes at room temperature. Thereafter, 50 μ of [³⁵S]GTPγS(0.6 nM) in Binding Buffer will be added to each well, followed byincubation on a shaker for 60 minutes at room temperature (again, inthis example, plates were covered with foil). The assay will then bestopped by spinning of the plates at 4000 RPM for 15 minutes at 22° C.The plates will then be aspirated with an 8 channel manifold and sealedwith plate covers. The plates will then be read on a Wallac 1450 usingsetting “Prot. #37” (as per manufacturer's instructions).

2. Adenylyl Cyclase Assay

A Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat. No.SMP004A) designed for cell-based assays can be modified for use withcrude plasma membranes. The Flash Plate wells can contain a scintillantcoating which also contains a specific antibody recognizing cAMP. ThecAMP generated in the wells can be quantitated by a direct competitionfor binding of radioactive cAMP tracer to the cAMP antibody. Thefollowing serves as a brief protocol for the measurement of changes incAMP levels in whole cells that express the receptors.

In certain embodiments, a modified Flash Plate™ Adenylyl Cyclase kit(New England Nuclear; Cat. No. SMP004A) is utilized for identificationof candidate compounds as, e.g., BRS-3 agonists in accordance with thefollowing protocol.

Cells transfected with a G protein-coupled receptor of the invention areharvested approximately three days after transfection. Membranes areprepared by homogenization of suspended cells in buffer containing 20 mMHEPES, pH 7.4 and 10 mM MgCl₂. Homogenization is performed on ice usinga Brinkman Polytron™ for approximately 10 seconds. The resultinghomogenate is centrifuged at 49,000×g for 15 minutes at 4° C. Theresulting pellet is then resuspended in buffer containing 20 mM HEPES,pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds, followed bycentrifugation at 49,000×g for 15 minutes at 4° C. The resulting pelletis then stored at −80° C. until utilized. On the day of directidentification screening, the membrane pellet is slowly thawed at roomtemperature, resuspended in buffer containing 20 mM HEPES, pH 7.4 and 10mM MgCl₂, to yield a final protein concentration of 0.60 mg/ml (theresuspended membranes are placed on ice until use).

cAMP standards and Detection Buffer (comprising 2 μCi of tracer {[¹²⁵I]cAMP (100 μl) to 11 ml Detection Buffer] are prepared and maintainedin accordance with the manufacturer's instructions. Assay Buffer wasprepared fresh for screening and contained 20 mM HEPES, pH 7.4, 10 mMMgCl₂, 20 mM phosphocreatine (Sigma), 0.1 units/ml creatinephosphokinase (Sigma), 50 μM GTP (Sigma), and 0.2 mM ATP (Sigma); AssayBuffer was then stored on ice until utilized.

Candidate compounds are added, preferably, to e.g. 96-well plate wells(3 μl/well; 12 μM final assay concentration), together with 401lMembrane Protein (30 μg/well) and 50 μl of Assay Buffer. This admixturewas then incubated for 30 minutes at room temperature, with gentleshaking.

Following the incubation, 100 μl of Detection Buffer is added to eachwell, followed by incubation for 2-24 hours. Plates are then counted ina Wallac MicroBeta™ plate reader using “Prot. #31” (as permanufacturer's instructions).

3. Reporter-Based Assays

a. CRE-Luc Reporter Assay 293 and 293T cells are plated-out on 96 wellplates at a density of 2×10⁴ cells per well and were transfected usingLipofectamine Reagent (BRL) the following day according to manufacturerinstructions. A DNA/lipid mixture is prepared for each 6-welltransfection as follows: 260 ng of plasmid DNA in 100 μl of DMEM isgently mixed with 2 μl of lipid in 100 μl of DMEM (the 260 ng of plasmidDNA consists of 200 ng of a 8xCRE-Luc reporter plasmid, 50 ng of pCMVcomprising a G protein-coupled receptor of the invention or pCMV alone,and 10 ng of a GPRS expression plasmid [GPRS in pcDNA3 (Invitrogen)].The 8XCRE-Luc reporter plasmid was prepared as follows: vectorSRIF-β-gal was obtained by cloning the rat somatostatin promoter(−71/+51) at BglV-HindIII site in the pβgal-Basic Vector (Clontech).Eight (8) copies of cAMP response element were obtained by PCR from anadenovirus template AdpCF126CCRE8 [see, Suzuki et al., Hum Gene Ther(1996) 7:1883-1893; the disclosure of which is herein incorporated byreference in its entirety) and cloned into the SRIF-β-gal vector at theKpn-BglV site, resulting in the 8xCRE-β-gal reporter vector. The8xCRE-Luc reporter plasmid was generated by replacing thebeta-galactosidase gene in the 8xCRE-β-gal reporter vector with theluciferase gene obtained from the pGL3-basic vector (Promega) at theHindIII-BamHI site. Following 30 min. incubation at room temperature,the DNA/lipid mixture is diluted with 400 μl of DMEM and 100 μof thediluted mixture is added to each well. 100 μl of DMEM with 10% FCS areadded to each well after a 4 hr incubation in a cell culture incubator.The following day the transfected cells are changed with 200 μl/well ofDMEM with 10% FCS. Eight (8) hours later, the wells are changed to 100μl/well of DMEM without phenol red, after one wash with PBS. Luciferaseactivity is measured the next day using the LucLite™ reporter gene assaykit (Packard) following manufacturer instructions and read on a 1450MicroBeta™ scintillation and luminescence counter (Wallac).

b. AP1 Reporter Assay (Gq-Associated Receptors)

A method to detect Gq stimulation depends on the known property ofGq-dependent phospholipase C to cause the activation of genes containingAP1 elements in their promoter. A Pathdetect™ AP-1 cis-Reporting System(Stratagene, Catalogue # 219073) can be utilized following the protocolset forth above with respect to the CREB reporter assay, except that thecomponents of the calcium phosphate precipitate were 410 ng pAP1-Luc, 80ng pCMV-receptor expression plasmid, and 20 ng CMV-SEAP.

c. SRF-Luc Reporter Assay (Gq-Associated Receptors)

One method to detect Gq stimulation depends on the known property ofGq-dependent phospholipase C to cause the activation of genes containingserum response factors in their promoter. A Pathdetect™SRF-Luc-Reporting System (Stratagene) can be utilized to assay for Gqcoupled activity in, e.g., COS7 cells. Cells are transfected with theplasmid components of the system and the indicated expression plasmidencoding endogenous or non-endogenous GPCR using a MammalianTransfection™ Kit (Stratagene, Catalogue #200285) according to themanufacturer's instructions. Briefly, 410 ng SRF-Luc, 80 ngpCMV-receptor expression plasmid and 20 ng CMV-SEAP (secreted alkalinephosphatase expression plasmid; alkaline phosphatase activity ismeasured in the media of transfected cells to control for variations intransfection efficiency between samples) are combined in a calciumphosphate precipitate as per the manufacturer's instructions. Half ofthe precipitate is equally distributed over 3 wells in a 96-well plate,kept on the cells in a serum free media for 24 hours. The last 5 hoursthe cells are incubated with, e.g. 1 μM, test compound. Cells are thenlysed and assayed for luciferase activity using a Luclite™ Kit (Packard,Cat. # 6016911) and “Trilux 1450 Microbeta” liquid scintillation andluminescence counter (Wallac) as per the manufacturer's instructions.The data can be analyzed using GraphPad Prism™ 2.0a (GraphPad SoftwareInc.).

4. Intracellular IP3 Accumulation Assay (Gq-Associated Receptors)

On day 1, cells to be transfected with a G protein-coupled receptor ofthe invention can be plated onto 24 well plates, usually 1×10⁵cells/well (although his number can be optimized). On day 2 cells can betransfected by first mixing 0.25 μg DNA (e.g., pCMV vector; pCMV vectorwith receptor cDNA, etc.) in 50 μl serum free DMEM/well and 2 μllipofectamine in 50 μl serum free DMEM/well. The solutions are gentlymixed and incubated for 15-30 min at room temperature. Cells are washedwith 0.5 ml PBS and 400 μl of serum free media is mixed with thetransfection media and added to the cells. The cells are then incubatedfor 3-4 hrs at 37° C./5% CO₂ and then the transfection media is removedand replaced with 1 ml/well of regular growth media. On day 3 the cellsare labeled with ³H-myo-inositol. Briefly, the media is removed and thecells are washed with 0.5 ml PBS. Then 0.5 ml inositol-free/serum freemedia (GIBCO BRL) is added/well with 0.25 μCi of ³H-myo-inositol/welland the cells are incubated for 16-18 hrs o/n at 37° C./5% CO₂. On Day 4the cells are washed with 0.5 ml PBS and 0.45 ml of assay medium isadded containing inositol-free/serum free media 10 μM pargyline 10 mMlithium chloride or 0.4 ml of assay medium and 50 μl of 10× TestCompound to final concentration of 10 μM. The cells are then incubatedfor 30 min at 37° C. The cells are then washed with 0.5 ml PBS and 200μl of fresh/ice cold stop solution (1M KOH; 18 mM Na-borate; 3.8 mMEDTA) is added/well. The solution is kept on ice for 5-10 min or untilcells were lysed and then neutralized by 200 μl of fresh/ice coldneutralization sol. (7.5% HCL). The lysate is then transferred into 1.5ml eppendorf tubes and 1 ml of chloroform/methanol (1:2) is added/tube.The solution is vortexed for 15 sec and the upper phase is applied to aBiorad AG1-X8™ anion exchange resin (100-200 mesh). Firstly, the resinis washed with water at 1:1.25 W/V and 0.9 ml of upper phase is loadedonto the column. The column is washed with 10 mls of 5 mM myo-inositoland 10 ml of 5 mM Na-borate/60 mM Na-formate. The inositol trisphosphates are eluted into scintillation vials containing 10 ml ofscintillation cocktail with 2 ml of 0.1 M formic acid/1 M ammoniumformate. The columns are regenerated by washing with 10 ml of 0.1 Mformic acid/3M ammonium formate and rinsed twice with dd H₂O and storedat 4° C. in water.

Example 4 Fluorometric Imaging Plate Reader (Flipr) Assay for theMeasurement of Intracellular Calcium Concentration (e.g., Gq-AssociatedReceptors)

Target Receptor (experimental) and pCMV (negative control) stablytransfected cells from respective clonal lines are seeded intopoly-D-lysine pretreated 96-well plates (Becton-Dickinson, #356640) at5.5×10⁴ cells/well with complete culture medium (DMEM with 10% FBS, 2 mML-glutamine, 1 mM sodium pyruvate) for assay the next day. To prepareFluo4-AM (Molecular Probe, #F14202) incubation buffer stock, 1 mgFluo4-AM is dissolved in 467 μl DMSO and 467 μl Pluoronic acid(Molecular Probe, #P3000) to give a 1 mM stock solution that can bestored at −20° C. for a month. Fluo4-AM is a fluorescent calciumindicator dye.

Candidate compounds are prepared in wash buffer (1×HBSS/2.5 mMProbenicid/20 mM HEPES at pH 7.4).

At the time of assay, culture medium is removed from the wells and thecells are loaded with 100 μl of 4 μM Fluo4-AM/2.5 mM Probenicid (Sigma,#P8761)/20 mM HEPES/complete medium at pH 7.4. Incubation at 37° C./5%CO₂ is allowed to proceed for 60 min.

After the 1 hr incubation, the Fluo4AM incubation buffer is removed andthe cells are washed 2× with 100 μl wash buffer. In each well is left100 μl wash buffer. The plate is returned to the incubator at 37° C./5%CO₂ for 60 min.

FLIPR (Fluorometric Imaging Plate Reader; Molecular Device) isprogrammed to add 50 μl candidate compound on the 30th second and torecord transient changes in intracellular calcium concentration ([Ca2+])evoked by the candidate compound for another 150 seconds. Totalfluorescence change counts are used to determine agonist activity usingthe FLIPR software. The instrument software normalizes the fluorescentreading to give equivalent initial readings at zero.

In some embodiments, the cells comprising Target Receptor furthercomprise Gα15, Gα16, or the chimeric Gq/Gi alpha unit.

Although the foregoing provides a FLIPR assay for agonist activity usingstably transfected cells, a person of ordinary skill in the art wouldreadily be able to modify the assay in order to characterize antagonistactivity. The person of ordinary skill in the art would also readilyappreciate that, alternatively, transiently transfected cells could beused.

Example 5 MAP Kinase Assay

MAP kinase (mitogen activated kinase) may be monitored to evaluatereceptor activation. MAP kinase can be detected by several approaches.One approach is based on an evaluation of the phosphorylation state,either unphosphorylated (inactive) or phosphorylated (active). Thephosphorylated protein has a slower mobility in SDS-PAGE and cantherefore be compared with the unstimulated protein using Westernblotting. Alternatively, antibodies specific for the phosphorylatedprotein are available (New England Biolabs) which can be used to detectan increase in the phosphorylated kinase. In either method, cells arestimulated with the test compound and then extracted with Laemmlibuffer. The soluble fraction is applied to an SDS-PAGE gel and proteinsare transferred electrophoretically to nitrocellulose or Immobilin.Immunoreactive bands are detected by standard Western blottingtechnique. Visible or chemiluminescent signals are recorded on film andmay be quantified by densitometry.

Another approach is based on evaluation of the MAP kinase activity via aphosphorylation assay. Cells are stimulated with the test compound and asoluble extract is prepared. The extract is incubated at 30° C. for 10min with gamma-³²P-ATP, an ATP regenerating system, and a specificsubstrate for MAP kinase such as phosphorylated heat and acid stableprotein regulated by insulin, or PHAS-I. The reaction is terminated bythe addition of H₃PO₄ and samples are transferred to ice. An aliquot isspotted onto Whatman P81 chromatography paper, which retains thephosphorylated protein. The chromatography paper is washed and countedfor ³²P is a liquid scintillation counter. Alternatively, the cellextract is incubated with gamma-³²P-ATP, an ATP regenerating system, andbiotinylated myelin basic protein bound by streptavidin to a filtersupport. The myelin basic protein is a substrate for activated MAPkinase. The phosphorylation reaction is carried out for 10 min at 30° C.The extract can then be aspirated through the filter, which retains, thephosphorylated myelin basic protein. The filter is washed and countedfor ³²P by liquid scintillation counting.

Example 6 Melanophore Technology

Melanophores are skin cells found in lower vertebrates. They containpigmented organelles termed melanosomes. Melanophores are able toredistribute these melanosomes along a microtubule network uponG-protein coupled receptor (GPCR) activation. The result of this pigmentmovement is an apparent lightening or darkening of the cells. Inmelanophores, the decreased levels of intracellular cAMP that resultfrom activation of a Gs-coupled receptor cause melanosomes to migrate tothe center of the cell, resulting in a dramatic lightening in color. IfcAMP levels are then raised, following activation of a Gs-coupledreceptor, the melanosomes are re-dispersed and the cells appear darkagain. The increased levels of diacylglycerol that result fromactivation of Gq-coupled receptors can also induce this re-dispersion.In addition, the technology is also suited to the study of certainreceptor tyrosine kinases. The response of the melanophores takes placewithin minutes of receptor activation and results in a simple, robustcolor change. The response can be easily detected using a conventionalabsorbance microplate reader or a modest video imaging system. Unlikeother skin cells, the melanophores derive from the neural crest andappear to express a full complement of signaling proteins. Inparticular, the cells express an extremely wide range of G-proteins andso are able to functionally express almost all GPCRs.

Melanophores can be utilized to identify compounds, including naturalligands, against GPCRs. This method can be conducted by introducing testcells of a pigment cell line capable of dispersing or aggregating theirpigment in response to a specific stimulus and expressing an exogenousclone coding for the GPCR. A stimulant, e.g., melatonin, sets an initialstate of pigment disposition wherein the pigment is aggregated withinthe test cells if activation of the GPCR induces pigment dispersion.However, stimulating the cell with a stimulant to set an initial stateof pigment disposition wherein the pigment is dispersed if activation ofthe GPCR induces pigment aggregation. The test cells are then contactedwith chemical compounds, and it is determined whether the pigmentdisposition in the cells changed from the initial state of pigmentdisposition. Dispersion of pigments cells due to the candidate compound,including but not limited to a ligand, coupling to the GPCR will appeardark on a petri dish, while aggregation of pigments cells will appearlight.

Materials and methods will be followed according to the disclosure ofU.S. Pat. No. 5,462,856 and U.S. Pat. No. 6,051,386. These patentdisclosures are hereby incorporated by reference in their entirety.

The cells are plated in e.g. 96-well plates (one receptor per plate). 48hours post-transfection, half of the cells on each plate are treatedwith 10 nM melatonin. Melatonin activates an endogenous G1-coupledreceptor in the melanophores and causes them to aggregate their pigment.The remaining half of the cells are transferred to serum-free medium0.7×L-15 (Gibco). After one hour, the cells in serum-free media remainin a pigment-dispersed state while the melatonin-treated cells are in apigment-aggregated state. At this point, the cells are treated with adose response of a test/candidate compound. If the plated GPCRs bind tothe test/candidate compound, the melanophores would be expected toundergo a color change in response to the compound. If the receptor wereeither a Gs or Gq coupled receptor, then the melatonin-aggregatedmelanophores would undergo pigment dispersion. In contrast, if thereceptor was a Gi-coupled receptor, then the pigment-dispersed cellswould be expected to undergo a dose-dependent pigment aggregation.

Example 7 Radiolabeled Compound

In certain embodiments, a compound known to be a ligand of a Gprotein-coupled receptor of the invention is radiolabeled. Aradiolabeled compound as described herein can be used in a screeningassay to identify/evaluate compounds. In general terms, a newlysynthesized or identified compound (i.e., test compound) can beevaluated for its ability to reduce binding of the radiolabeled knownligand to the receptor, by its ability to reduce formation of thecomplex between the radiolabeled known ligand and the receptor. Suitableradionuclides that may be incorporated in compounds of the presentinvention include but are not limited to ³H (also written as T), ¹¹C,¹⁴C, ¹⁸F, ¹²⁵I, ⁸²Br, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ¹⁵O, ¹³N, ³⁵Sand ⁷⁷Br. Compounds that incorporate ³H, ¹⁴C, ¹²⁵I, ¹³¹I, ³⁵S or ⁸²Brwill generally be most useful.

It is understood that a “radiolabelled” compound” is a compound that hasincorporated at least one radionuclide. In some embodiments, theradionuclide is selected from the group consisting of ³H, ¹⁴C, ¹²⁵I, ³⁵Sand ⁸²Br. In some embodiments, the radionuclide ³H or ¹⁴C. Moreover, itshould be understood that all of the atoms represented in the compoundsknown to be ligands of a G protein-coupled receptor of the invention canbe either the most commonly occurring isotope of such atoms or the morescarce radioisotope or nonradioactive isotope.

Synthetic methods for incorporating radioisotopes into organic compoundsincluding those applicable to those compounds known to be ligands of a Gprotein-coupled receptor of the invention are well known in the art andinclude incorporating activity levels of tritium into target moleculesinclude: A. Catalytic Reduction with Tritium Gas—This procedure normallyyields high specific activity products and requires halogenated orunsaturated precursors. B. Reduction with Sodium Borohydride [³H]Thisprocedure is rather inexpensive and requires precursors containingreducible functional groups such as aldehydes, ketones, lactones,esters, and the like. C. Reduction with Lithium Aluminum Hydride[³H]—This procedure offers products at almost theoretical specificactivities. It also requires precursors containing reducible functionalgroups such as aldehydes, ketones, lactones, esters, and the like. D.Tritium Gas Exposure Labeling—This procedure involves exposingprecursors containing exchangeable protons to tritium gas in thepresence of a suitable catalyst. E. N-Methylation using Methyl Iodide[³H]—This procedure is usually employed to prepare O-methyl or N-methyl(³H) products by treating appropriate precursors with high specificactivity methyl iodide (³H). This method in general allows for highspecific activity, such as about 80-87 Ci/mmol.

Synthetic methods for incorporating activity levels of ¹²⁵I into targetmolecules include: A. Sandmeyer and like reactions—This proceduretransforms an aryl or heteroaryl amine into a diazonium salt, such as atetrafluoroborate salt, and subsequently to ¹²⁵I labelled compound usingNa¹²⁵. A represented procedure was reported by Zhu, D.-G. and co-workersin J. Org. Chem. 2002, 67, 943-948. B. Ortho ¹²⁵Iodination ofphenols—This procedure allows for the incorporation of ¹²⁵I at the orthoposition of a phenol as reported by Collier, T. L. and co-workers in J.Labelled Compd Radiopharm. 1999, 42, S264-S266. C. Aryl and heteroarylbromide exchange with ¹²⁵I—This method is generally a two step process.The first step is the conversion of the aryl or heteroaryl bromide tothe corresponding tri-alkyltin intermediate using for example, a Pdcatalyzed reaction [i.e. Pd(Ph₃P)₄] or through an aryl or heteroaryllithium, in the presence of a tri-alkyltinhalide or hexaalkylditin[e.g., (CH₃)₃SnSn(CH₃)₃]. A represented procedure was reported by Bas,M.-D. and co-workers in J. Labelled Compd Radiophart. 2001, 44,S280-S282.

The foregoing techniques are intended to be illustrative and notlimiting. Other techniques for radiolabeling a compound known to be aligand of a G protein-coupled receptor of the invention are well knownto the skilled artisan.

Example 8 Receptor Binding Assay

A test compound can be evaluated for its ability to reduce formation ofthe complex between a compound known to be a ligand of a Gprotein-coupled receptor of the invention and the receptor. In certainembodiments, the known ligand is radiolabeled. The radiolabeled knownligand can be used in a screening assay to identify/evaluate compounds.In general terms, a newly synthesized or identified compound (I.e., testcompound) can be evaluated for its ability to reduce binding of theradiolabeled known ligand to the receptor, by its ability to reduceformation of the complex between the radiolabeled known ligand and thereceptor.

Assay Protocol for Detecting the Complex Between a Compound Known to bea Ligand of a G Protein-Coupled Receptor of the Invention and theReceptor

A. Preparation of the Receptor

293 cells are transiently transfected with 10 ug expression vectorcomprising a polynucleotide encoding a G protein-coupled receptor of theinvention using 60 ul Lipofectamine (per 15-cm dish). The transientlytransfected cells are grown in the dish for 24 hours (75% confluency)with a media change and removed with 10 ml/dish of Hepes-EDTA buffer (20mM Hepes+10 mM EDTA, pH 7.4). The cells are then centrifuged in aBeckman Coulter centrifuge for 20 minutes, 17,000 rpm (JA-25.50 rotor).Subsequently, the pellet is resuspended in 20 mM Hepes+1 mM EDTA, pH 7.4and homogenized with a 50-ml Dounce homogenizer and again centrifuged.After removing the supernatant, the pellets are stored at −80° C., untilused in binding assay. When used in the assay, membranes are thawed onice for 20 minutes and then 10 mL of incubation buffer (20 mM Hepes, 1mM MgCl₂, 100 mM NaCl, pH 7.4) added. The membranes are then vortexed toresuspend the crude membrane pellet and homogenized with a BrinkmannPT-3100 Polytron homogenizer for 15 seconds at setting 6. Theconcentration of membrane protein is determined using the BRL Bradfordprotein assay.

B. Binding Assay

For total binding, a total volume of 50 ul of appropriately dilutedmembranes (diluted in assay buffer containing 50 mM Tris HCl (pH 7.4),10 mM MgCl₂, and 1 mM EDTA; 5-50 ug protein) is added to 96-wellpolyproylene microtiter plates followed by addition of 100 ul of assaybuffer and 50 ul of a radiolabeled known ligand. For nonspecificbinding, 50 ul of assay buffer is added instead of 100 ul and anadditional 50 ul of 10 uM said known ligand which is not radiolabeled isadded before 50 ul of said radiolabeled known ligand is added. Platesare then incubated at room temperature for 60-120 minutes. The bindingreaction is terminated by filtering assay plates through a MicroplateDevices GF/C Unifilter filtration plate with a Brandell 96-well plateharvester followed by washing with cold 50 mM Tris HCl, pH 7.4containing 0.9% NaCl. Then, the bottom of the filtration plate aresealed, 50 ul of Optiphase Supermix is added to each well, the top ofthe plates are sealed, and plates are counted in a Trilux MicroBetascintillation counter. For determining whether less of the complexbetween said radiolabeled known ligand and said receptor is formed inthe presence of a test compound, instead of adding 100 ul of assaybuffer, 100 ul of appropriately diluted said test compound is added toappropriate wells followed by addition of 50 ul of said radiolabledknown ligand.

A level of specific binding of the radiolabled known ligand in thepresence of the test compound less than a level of specific binding ofthe radiolabeled known ligand in the absence of the test compound isindicative of less of the complex between said radiolabeled known ligandand said receptor being formed in the presence of the test compound thanin the absence of the test compound.

Example 9 BRS-3 Increases Intracellular IP3 Accumulation

COS-7 cells were transiently transfected with pCMV expression vectorcontaining cDNA encoding endogenous human BRS-3 or with pCMV vectoralone. Intracellular IP3 accumulation was read out as accumulation oftotal inositol phosphates.

COS-7 cells were plated at 10,000 cells per well in a 96-well plate andallowed to attach overnight. The COS-7 cells were then transfected intriplicate with 0.5 or 1.3 ng/well BRS-3/pCMV or with 13 ng/well emptypCMV, using Lipofectamine™ 2000 (Invitrogen #11668-027). After about 15h, the transfected COS-7 cells were returned to complete medium (DMEMcontaining 10% FBS, 1% L-glutamine, and 1.5 g/L sodium bicarbonate) andcell culture was continued at 37° C. for about 8 hours.

The COS-7 cells were used in IP3 assay about 24 h post-transfection asdescribed here. The complete medium was replaced with 100 μlinositol-free medium (Invitrogen/Gibco formula 02-5092EA; DMEMcontaining D-glucose, L-glutamine, phenol red, and pyridoxine HCl, andwithout inositol, sodium bicarbonate, and sodium pyruvate) supplementedwith 1.5 g/L sodium bicarbonate and 4 □Ci/ml [³H]myo-inositol (PerkinElmer Life Sciences), and the cells were allowed to incubate for about15 h at 37° C. The medium was then removed by aspiration and replacedwith IP3 medium (inositol-free medium as above supplemented with 10 μMpargyline and 10 mM lithium chloride), and the cells were incubated for3 hours at 37° C. (To screen a test compound as a BRS-3 agonist, thetest compound would be included in this 3 h incubation.) Followingincubation, the medium was removed by aspiration and replaced withbuffer containing ice cold 0.1M formic acid. The plates were then frozenovernight at −80° C. to achieve complete cell lysis following an initial30 min incubation on dry ice.

Following complete cell lysis, the assay plates were thawed in a 37° C.oven. The thawed contents were then transferred to 96-well filter plates(Nillipore, Multiscreen) pre-loaded with resin (Biorad, AG1-X8 100-200mesh, formate form). The plate was filtered using a vacuum manifold andthe resin was washed multiple times with water. An elution buffer wasthen applied (200 μl, 1.0M ammonium formate/0.1M formic acid) and theresulting eluent was collected, under vacuum, in a 96-well collectionplate. Aliquots of the eluent (200 μl) were transferred to scintillationvials containing 4 ml scintillation fluid and counted on a scintillationcounter (Perkin Elmer Life Sciences, Optiphase Supermix or Hi-Safe 3).

BRS-3 was found to exhibit a detectable level of constitutive activityand to increase the level of 1P3 accumulation in COS-7 cells. SeeFIG. 1. Analogous results were obtained using 293 cells (not shown).

Example 10 RT-PCR Analysis OF BRS-3 Expression in GLUTag Cells

RT-PCR was used to determine BRS-3 expression in GLUTag cells. GLUTag isa GLP-1 producing mouse enteroendocrine L-cell line [Brubaker et al.,Endocrinology (1998) 139:4108-4114].

BRS-3 expression in GLUTag cells was evaluated by RT-PCR using thefollowing primers:

(SEQ ID NO: 5; sense) 5′-TCCCGCTCTCGATTATCTCTGTCT-3′ and (SEQ ID NO: 6;antisense) 5′-TCCTCTCCCTTCTTGGCACTACTG-3′.PCR primers SEQ ID NO:5 and SEQ ID NO:6 were chosen so as to amplifyBRS-3 across an intron, such that the amplification product for cDNA(508 bp) is easily distinguishable from that for genomic DNA.

Total RNA was isolated from GLUTag cells using Trizol Reagent purchasedfrom Invitrogen. The cDNA synthesis was performed by using a cDNASynthesis kit from Bio-Rad (iScript™). PCR was performed using TaqSupermix (Invitrogen) in a 50 μl reaction mixture containing 2 μl cDNAand 100 ng of each primer. Control reactions included a no-templatecontrol and a mouse genomic DNA template control. Samples were placed ina thermal cycler and incubated at 94C.° for 5 minutes, followed by 30cycles of [30 seconds at 94C.°, 30 seconds at 57.5C.°, and 1 minute at72C.°], and a final incubation of 72° C. for 10 minutes. 5 μl of eachPCR reaction product was then analyzed on a 1%/TAE Agarose Gel.

GLUTag cells were found to express BRS-3. See FIG. 2.

Example 11 Effect OF BRS-3 Agonist on Intracellular IP3 Accumulation inGLUTag Cells

GLUTag is a GLP-1 producing mouse enteroendocrine L-cell line [Brubakeret al., Endocrinology (1998) 139:4108-4114]. The effect of BRS-3 agoniston the level of intracellular IP3 accumulation in GLUTag cells isdetermined.

IP3 assay in GluTag cells is carried out as described in Example 9,supra. The GLUTag cells are incubated with or without BRS-3 agonistduring the 3 h incubation in IP3 medium. In certain embodiments, theBRS-3 agonist is selected from the left column of Table B.

BRS-3 agonist is found to increase intracellular IP3 accumulation inGLUTag cells.

Example 12 Effect OF BRS-3 Agonist on Stimulation of GLP-1 Secretion inGLUTag Cells

GLUTag is a GLP-1 producing mouse enteroendocrine L-cell line [Brubakeret al., Endocrinology (1998) 139:4108-4114]. The effect of BRS-3 agoniston stimulation of GLP-1 secretion in GLUTag cells is determined.

GLUTag cells are plated in 24-well plates on day one in complete culturemedium (DMEM/10% FBS). On day two the culture medium is replaced with alow glucose medium (DMEM/3mM Glucose/10%FBS). On day three cells arewashed twice with 1×PBS. The washed GLUTag cells are stimulated withBRS-3 agonist at various concentrations (from about 1 nM to about 10 μM,by way of illustration and not limitation) or with forskolin (1 μl) as apositive control in serum free DMEM with 15 mM glucose for one hour at37° C. and 5% CO₂ in a tissue culture incubator. The supernatants arethen collected and clarified by centrifugation at 500 g and 4° C. for 5minutes. GLP-1 released into the supernatant is determined by ELISAusing reagents purchased from LINCO Research Laboratory [Glucagon-LikePeptide-1 (Active) ELISA Kit. Cat. # EGLP-35K].

GLUTag cells are found to secrete GLP-1 when stimulated with BRS-3agonist.

Example 13 Effect of BRS-3 Agonist and DPP-IV Inhibitor in Lowering anElevated Blood Glucose Level in Oral Glucose Tolerance Test (OGTT) inMice

Oral glucose tolerance test (OGTT) in mice is carried out as describedhere.

In mouse oGTT assay, a therapeutically effective amount is typicallythat amount of drug which will create an AUC inhibition above 30%,whereas a therapeutically ineffective amount is typically an amount ofdrug which will create an AUC inhibition less than or equal to 30%.

Overnight fasted mice (n=6 mice per treatment) are administered via oralgavage with vehicle (PET), a BRS-3 agonist [e.g., at a dose betweenabout 0.1 mkg to about 1 mkg (milligram compound per kilogram of bodyweight) which is therapeutically effective when used alone], a DPP-IVinhibitor [e.g., at a dose between about 0.1 mkg to about 1 mkg which istherapeutically ineffective when used alone], or a combination of theBRS-3 agonist and the DPP-IV inhibitor (e.g., at the foregoingtherapeutically ineffective doses). Thirty minutes later, a glucosebolus (3 gram/kg) is then delivered per orally. Plasma glucose levelsare determined at about 20 min intervals over a two hour period usingblood (˜5 μl) collected from tail nick and a glucose meter. Glycemicexcursion curve is graphed based on data from 6 mice and is given inmean values +/−SEM. Area Under Curve (AUC) of the glycemic excursion iscalculated for each mouse and AUC inhibition (%) is determined.

The combination of BRS-3 agonist and DPP-IV inhibitor is found toproduce a synergistic AUC inhibition compared to that of BRS-3 agonistalone or DPP-IV inhibitor alone.

The combination of an amount of a BRS-3 agonist and a DPP-IV inhibitoris found to produce an AUC inhibition greater than the inhibition givenby the amount of the BRS-3 agonist alone and the inhibition given by theamount of the DPP-IV inhibitor alone.

Example 14 Combination of BRS-3 Agonist and DPP-IV Inhibitor forTreating or Preventing Diabetes and Conditions Related Thereto

A BRS-3 agonist in accordance with the present invention is selected. ADPP-IV inhibitor in accordance with the present invention is selected.

Titration of the BRS-3 agonist with respect to percent inhibition ofArea Under Curve (AUC) in mouse oral glucose tolerance test (oGTT) isdetermined across a dose range from about 0.01 mkg (milligram compoundper kilogram of body weight) to about 100 mkg. See Example 13, supra. Adose of the BRS-3 agonist producing an AUC inhibition of glycemicexcursion of about 15-20% is chosen. Typically, a dose of BRS-3 agonistproducing an AUC inhibition 30% or less is therapeutically ineffectivein this mouse model.

Titration of the DPP-IV inhibitor with respect to percent inhibition ofArea Under Curve (AUC) in mouse oral glucose tolerance test (oGTT) isdetermined across a dose range from about 0.01 mkg (milligram compoundper kilogram of body weight) to about 100 mkg. See Example 13, supra. Adose of the DPP-IV inhibitor producing an AUC inhibition of glycemicexcursion of about 15-20% is chosen. Typically, a dose of DPP-IVinhibitor producing an AUC inhibition 30% or less is therapeuticallyineffective in this mouse model.

The AUC inhibition of glycemic excursion produced by the combination ofthe chosen dose of the BRS-3 agonist and the chosen dose of the DPP-IVinhibitor is determined in mouse oGTT assay. Therapeutic efficacy of thecombination of the BRS-3 agonist and the DPP-IV inhibitor is determined.Typically, an amount of the combination producing an AUC inhibitionabove 30% is therapeutically effective in this mouse model. Synergismbetween the BRS-3 agonist and the DPP-IV inhibitor is determined.

Data obtained from this mouse model can be used to formulate a range ofdoseage for use in humans. In general, one skilled in the artunderstands how to extrapolate in vivo data obtained in an animal modelsystem to another, such as a human. A combination of BRS-3 agonist andDPP-IV inhibitor in accordance with the present invention is useful intreating or preventing diabetes and conditions related thereto.

It is understood that the foregoing is intended to be illustrative andnot limiting.

Example 15 Effect of BRS-3 Agonist and DPP-IV Inhibitor in Increasing aBlood GLP-1 Level After Glucose Challenge in Mice

C57blk/6 male mice (8 weeks of age) are fasted for 18 hours, andrandomly assigned into twelve groups with n=6 for each group. Mice areadministered per orally with vehicle (PET), a BRS-3 agonist [e.g., at adose between about 0.1 mkg to about 1 mkg (milligram compound perkilogram of body weight) which is therapeutically effective when usedalone], a DPP-IV inhibitor [e.g., at a dose between about 0.1 mkg toabout 1 mkg which is therapeutically ineffective when used alone], or acombination of the BRS-3 agonist and the DPP-IV inhibitor (e.g., at theforegoing therapeutically ineffective doses). Thirty minutes aftertreatment, a glucose bolus at 3 g/kg is delivered per orally, and plasmais collected at 0 minute (no glucose bolus), and at 2 minutes and 5minutes after glucose bolus. Plasma GLP-1 levels are determined by usinga GLP-1 ELISA kit purchased from Linco Research Laboratory[Glucagon-Like Peptide-1 (Active) ELISA kit, Catalog #EGLP-35K].

Administration of a BRS-3 agonist together with a DPP-V inhibitor isfound to produce a synergistic effect in increasing a blood GLP-1 level.

Administration of an amount of a BRS-3 agonist in combination with anamount of a DPP-IV inhibitor is found to produce an effect in increasinga blood GLP-1 level greater than the effect given by the amount of theBRS-3 agonist alone and the effect given by the amount of the DPP-IVinhibitor alone.

Example 16 Yeast Reporter Assay for BRS-3 Agonist Activity

The yeast cell-based reporter assays have previously been described inthe literature (e.g., see Miret et al, J Biol Chem (2002) 277:6881-6887;Campbell et al, Bioorg Med Chem Lett (1999) 9:2413-2418; King et al,Science (1990) 250:121-123; WO 99/14344; WO 00/12704; and U.S. Pat. No.6,100,042). Briefly, yeast cells have been engineered such that theendogenous yeast G-alpha (GPA1) has been deleted and replaced withG-protein chimeras constructed using multiple techniques. Additionally,the endogenous yeast alpha-cell GPCR, Step 3 has been deleted to allowfor a homologous expression of a mammalian GPCR of choice. In the yeast,elements of the pheromone signaling transduction pathway, which areconserved in eukaryotic cells (for example, the mitogen-activatedprotein kinase pathway), drive the expression of Fus1. By placingβ-galactosidase (LacZ under the control of the Fus1 promoter (Fus1p), asystem has been developed whereby receptor activation leads to anenzymatic readout.

Yeast cells are transformed by an adaptation of the lithium acetatemethod described by Agatep et al (Agatep et al, 1998, Transformation ofSaccharomyces cerevisiae by the lithium acetate/single-stranded carrierDNA/polyethylene glycol (LiAc/ss-DNA/PEG) protocol. Technical TipsOnline, Trends Journals, Elsevier). Briefly, yeast cells are grownovernight on yeast tryptone plates (YT). Carrier single-stranded DNA (10μg), 2 μg of each of two Fuslp-LacZ reporter plasmids (one with URAselection marker and one with TRP), 2 μg of BRS-3 (e.g., human receptor)in yeast expression vector (2 μg origin of replication) and a lithiumacetate/polyethylene glycol/TE buffer is pipetted into an Eppendorftube. The yeast expression plasmid containing the receptor/no receptorcontrol has a LEU marker. Yeast cells are inoculated into this mixtureand the reaction proceeds at 30° C. for 60 min. The yeast cells are thenheat-shocked at 42° C. for 15 min. The cells are then washed and spreadon selection plates. The selection plates are synthetic defined yeastmedia minus LEU, URA and TRP(SD-LUT). After incubating at 30° C. for 2-3days, colonies that grow on the selection plates are then tested in theLacZ assay.

In order to perform fluorimetric enzyme assays for β-galactosidase,yeast cells carrying the subject BRS-3 receptor are grown overnight inliquid SD-LUT medium to an unsaturated concentration (i.e. the cells arestill dividing and have not yet reached stationary phase). They arediluted in fresh medium to an optimal assay concentration and 90 μl ofyeast cells are added to 96-well black polystyrene plates (Costar). Testcompounds, dissolved in DMSO and diluted in a 10% DMSO solution to 10×concentration, are added to the plates and the plates placed at 30° C.for 4 h. After 4 h, the substrate for the β-galactosidase is added toeach well. In these experiments, Fluorescein di (β-galactopyranoside) isused (FDG), a substrate for the enzyme that releases fluorescein,allowing a fluorimetric read-out. 20 μl per well of 500 μM FDG/2.5%Triton X100 is added (the detergent is necessary to render the cellspermeable). After incubation of the cells with the substrate for 60 min,20 μl per well of 1M sodium carbonate is added to terminate the reactionand enhance the fluorescent signal. The plates are then read in afluorimeter at 485/535 nm.

An increase in fluorescent signal pigment dispersion inBRS-3-transformed yeast cells over that in yeast cells transformed withempty vector is indicative of a test compound being a compound thatstimulates BRS-3 receptor functionality.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be understood that the practice of the invention encompasses all ofthe usual variations, adaptions, or modifications, as come within thescope of the following claims and its equivalents.

1. A method for identifying a GLP-1 secretagogue or a compound usefulfor treating a condition ameliorated by increasing a GLP-1 level,comprising the steps of: (a) contacting a test compound with an isolatedhost cell or with a membrane of a host cell that expresses a Gprotein-coupled receptor, wherein the G protein-coupled receptorcomprises an amino acid sequence selected from the group consisting of:(i) amino acids 1-335 of SEQ ID NO:2; (ii) amino acids 1-399 of SEQ IDNO:2; (iii) amino acids 2-335 of SEQ ID NO:2; (iv) amino acids 2-399 ofSEQ ID NO:2; (v) the amino acid sequence of a G protein-coupled receptorencoded by a polynucleotide comprising a nucleotide sequence, thenucleotide sequence being the sequence obtainable by a processcomprising performing polymerase chain reaction (PCR) on a human DNAsample using specific primers SEQ ID NO:3 and SEQ ID NO:4; and (vi) anamino acid sequence that is at least 95% identical to SEQ ID NO: 2; and(b) determining the ability of the test compound to stimulatefunctionality of the G protein-coupled receptor; (c) contacting a testcompound which stimulates functionality of the G protein-coupledreceptor in step (b) in vitro with a mammalian enteroendocrine cell; and(d) determining whether the test compound stimulates GLP-1 secretionfrom the mammalian enteroendocrine cell; wherein the ability of the testcompound to stimulate GLP-1 secretion from the mammalian enteroendocrinecell is indicative of the test compound being a GLP-1 secretagogue or acompound useful for treating a condition ameliorated by increasing aGLP-1 level.
 2. The method of claim 1, wherein the method is foridentifying a GLP-1 secretagogue.
 3. The method of claim 1, wherein thecondition ameliorated by increasing a GLP-1 level is selected from thegroup consisting of diabetes, a condition related to diabetes,myocardial infarction, learning impairment, memory impairment, and aneurodegenerative disorder.
 4. The method of claim 1, wherein the hostcell comprises an expression vector, the expression vector comprising apolynucleotide encoding the G protein-coupled receptor.
 5. The method ofclaim 1, wherein the determining the ability of the test compound tostimulate functionality of the G protein-coupled receptor is through themeasurement of the level of a second messenger.
 6. The method of claim5, wherein the second messenger is selected from the group consisting ofcyclic AMP (cAMP), cyclic GMP (cGMP), inositol 1,4,5-triphosphate (IP₃),diacylglycerol (DAG), MAP kinase activity, MAPK/ERK kinase kinase-1(MEKK1) activity, and Ca²⁺.
 7. The method of claim 6, wherein a level ofIP₃ is increased.
 8. The method of claim 1, wherein the determining theability of the test compound to stimulate functionality of the Gprotein-coupled receptor is through the use of a Melanophore assay, orthrough the measurement of GTPγS binding to a membrane comprising theGPCR.
 9. The method of claim 1, wherein the mammalian enteroendocrinecell is a GLUTag enteroendocrine L-cell line.
 10. A method foridentifying a GLP-1 secretagogue or a compound useful for treating acondition ameliorated by increasing a GLP-1 level, comprising the stepsof: (a) contacting a test compound with an isolated host cell or with amembrane of a host cell that expresses a G protein-coupled receptor,wherein the G protein-coupled receptor comprises an amino acid sequenceselected from the group consisting of: (i) amino acids 1-335 of SEQ IDNO:2; (ii) amino acids 1-399 of SEQ ID NO:2; (iii) amino acids 2-335 ofSEQ ID NO:2; (iv) amino acids 2-399 of SEQ ID NO:2; (v) the amino acidsequence of a G protein-coupled receptor encoded by a polynucleotidecomprising a nucleotide sequence, the nucleotide sequence being thesequence obtainable by a process comprising performing polymerase chainreaction (PCR) on a human DNA sample using specific primers SEQ ID NO:3and SEQ ID NO:4; and (vi) an amino acid sequence that is at least 95%identical to SEQ ID NO: 2; and (b) determining the ability of the testcompound to stimulate functionality of the G protein-coupled receptor;(c) determining the GLP-1 level in a biological sample obtained from amammal, the mammal having been administered with a test compound whichstimulates functionality of the G protein-coupled receptor in step (b);wherein the ability of the test compound to increase a GLP-1 level inthe mammal is indicative of the test compound being a GLP-1 secretagogueor a compound useful for treating a condition ameliorated by increasinga GLP-1 level.
 11. The method of claim 10, wherein the method is foridentifying a GLP-1 secretagogue.
 12. The method of claim 10, whereinthe condition ameliorated by increasing a GLP-1 level is selected fromthe group consisting of diabetes, a condition related to diabetes,myocardial infarction, learning impairment, memory impairment, and aneurodegenerative disorder.
 13. The method of claim 10, wherein themammal is a non-human mammal.
 14. The method of claim 10, wherein themammal is a human.
 15. The method of claim 10, wherein the host cellcomprises an expression vector, the expression vector comprising apolynucleotide encoding the G protein-coupled receptor.
 16. The methodof claim 10, wherein the determining the ability of the test compound tostimulate functionality of the G protein-coupled receptor is through themeasurement of the level of a second messenger.
 17. The method of claim16, wherein the second messenger is selected from the group consistingof cyclic AMP (cAMP), cyclic GMP (cGMP), inositol 1,4,5-triphosphate(IP₃), diacylglycerol (DAG), MAP kinase activity, MAPK/ERK kinasekinase-1 (MEKK1) activity, and Ca²⁺.
 18. The method of claim 17, whereina level of IP₃ is increased.
 19. The method of claim 10, wherein thedetermining the ability of the test compound to stimulate functionalityof the G protein-coupled receptor is through the use of a Melanophoreassay, or through the measurement of GTPγS binding to a membranecomprising the GPCR.
 20. A method for identifying a GLP-1 secretagogueor a compound useful for treating a condition ameliorated by increasinga GLP-1 level, comprising the steps of: (a) contacting a Gprotein-coupled receptor with an optionally labeled known ligand to thereceptor in the presence or absence of a test compound, wherein the Gprotein-coupled receptor comprises an amino acid sequence selected fromthe group consisting of: (i) amino acids 1-335 of SEQ ID NO:2; (ii)amino acids 1-399 of SEQ ID NO:2; (iii) amino acids 2-335 of SEQ IDNO:2; (iv) amino acids 2-399 of SEQ ID NO:2; (v) the amino acid sequenceof a G protein-coupled receptor encoded by a polynucleotide comprising anucleotide sequence, the nucleotide sequence being the sequenceobtainable by a process comprising performing PCR on a human DNA sampleusing specific primers SEQ ID NO:3 and SEQ ID NO:4; and (vi) an aminoacid sequence that is at least 95% identical to SEQ ID NO: 2; and (b)detecting the complex between the known ligand and the G protein-coupledreceptor; and (c) determining whether less of the complex is formed inthe presence of the test compound than in the absence of the testcompound; (d) contacting a test compound in the presence of which lessof the complex is formed in step (c) in vitro with a mammalianenteroendocrine cell; and (e) determining whether the test compoundstimulates GLP-1 secretion from the mammalian enteroendocrine cell;wherein the ability of the test compound to stimulate GLP-1 secretionfrom the mammalian enteroendocrine cell is indicative of the testcompound being a GLP-1 secretagogue or a compound useful for treating acondition ameliorated by increasing a GLP-1 level.
 21. The method ofclaim 20, wherein the method is for identifying a GLP-1 secretagogue.22. The method of claim 20, wherein the condition ameliorated byincreasing a GLP-1 level is selected from the group consisting ofdiabetes, a condition related to diabetes, myocardial infarction,learning impairment, memory impairment, and a neurodegenerativedisorder.
 23. The method of claim 20, wherein the receptor isrecombinant.
 24. The method of claim 20, wherein the mammalianenteroendocrine cell is a GLUTag enteroendocrine L-cell line.
 25. Amethod for identifying a GLP-1 secretagogue or a compound useful fortreating a condition ameliorated by increasing a GLP-1 level, comprisingthe steps of: (a) contacting a G protein-coupled receptor with anoptionally labeled known ligand to the receptor in the presence orabsence of a test compound, wherein the G protein-coupled receptorcomprises an amino acid sequence selected from the group consisting of:(i) amino acids 1-335 of SEQ ID NO:2; (ii) amino acids 1-399 of SEQ IDNO:2; (iii) amino acids 2-335 of SEQ ID NO:2; (iv) amino acids 2-399 ofSEQ ID NO:2; (v) the amino acid sequence of a G protein-coupled receptorencoded by a polynucleotide comprising a nucleotide sequence, thenucleotide sequence being the sequence obtainable by a processcomprising performing PCR on a human DNA sample using specific primersSEQ ID NO:3 and SEQ ID NO:4; and (b) detecting the complex between theknown ligand and the G protein-coupled receptor; and (c) determiningwhether less of the complex is formed in the presence of the testcompound than in the absence of the test compound; (d) determining theGLP-1 level in a biological sample obtained from a mammal, the mammalhaving been administered with a test compound in the presence of whichless of the complex is formed in step (c); wherein the ability of thetest compound to increase a GLP-1 level in the mammal is indicative ofthe test compound being a GLP-1 secretagogue or a compound useful fortreating a condition ameliorated by increasing a GLP-1 level.
 26. Themethod of claim 25, wherein the method is for identifying a GLP-1secretagogue.
 27. The method of claim 25, wherein the conditionameliorated by increasing a GLP-1 level is selected from the groupconsisting of diabetes, a condition related to diabetes, myocardialinfarction, learning impairment, memory impairment, and aneurodegenerative disorder.
 28. The method of claim 25, wherein themammal is a non-human mammal.
 29. The method of claim 25, wherein themammal is a human.
 30. The method of claim 25, wherein the receptor isrecombinant.